Review Article

The role of TREX in gene expression and diseaseCatherine G. Heath, Nicolas Viphakone and Stuart A. WilsonDepartment of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, U.K.

Correspondence: Stuart A. Wilson (stuart.wilson@sheffield.ac.uk)

TRanscription and EXport (TREX) is a conserved multisubunit complex essential forembryogenesis, organogenesis and cellular differentiation throughout life. By linking tran-scription, mRNA processing and export together, it exerts a physiologically vital role inthe gene expression pathway. In addition, this complex prevents DNA damage and regu-lates the cell cycle by ensuring optimal gene expression. As the extent of TREX activity inviral infections, amyotrophic lateral sclerosis and cancer emerges, the need for a greaterunderstanding of TREX function becomes evident. A complete elucidation of the com-position, function and interactions of the complex will provide the framework for under-standing the molecular basis for a variety of diseases. This review details the knowncomposition of TREX, how it is regulated and its cellular functions with an emphasis onmammalian systems.

IntroductionProtein-coding genes transcribed by RNA polymerase II (Pol II) give rise to RNA molecules termedpre-mRNAs. During and/or after their synthesis, these precursors undergo a series of three main process-ing steps. Their 50 end receives an m7G cap structure, shortly after the initiation of transcription, whichprotects the nascent pre-mRNA from degradation and plays a role in further stages of mRNA maturation[1]. The introns are excised during splicing which generally occurs co-transcriptionally, althoughapproximately 20% of splicing events take place following transcription [2]. The 30-end of the pre-mRNAis processed by endonucleolytic cleavage and polyadenylation, leaving the transcript with a polyadenosine(poly(A)) tail [3]. Following these processing events, the mRNA is exported from the nucleus throughthe nuclear pore complex to the cytoplasm where it can be translated into proteins (Figure 1).From its site of synthesis until it reaches the nuclear pore, the future mRNA associates with numer-

ous proteins to form a messenger ribonucleoprotein (mRNP). Throughout its nuclear odyssey, themRNP composition is modified by the various remodeling events that accompany each maturationstage (Figure 1). The completion of each processing step results not only in a modified RNA moleculebut also in the association of a specific set of proteins with the RNA. These two hallmarks of amature mRNA are monitored by quality control mechanisms that trigger the degradation of defectivemRNAs either in the nucleus by the nuclear exosome [4] or in the cytoplasm via the NMD pathway[5]. The expression of a functional protein thus relies on a properly synthesized and processed mRNAtranscript packaged with the appropriate group of proteins.A key player in mRNP biogenesis and maturation is the TRanscription and EXport (TREX)

complex. TREX is conserved across a wide range of organisms including Saccharomyces cerevisiae,Drosophila, Arabidopsis, Xenopus and humans [6–10], indicating its key physiological importance.Components of TREX are functionally linked to each mRNA processing step, and this is reflected bytheir physical association with protein complexes such as the 50 cap-binding complex (CBC), the exonjunction complex (EJC) and 30-end processing factors [11–13] (Figure 1). Therefore, TREX probablyacts as an interface for these various processes to help maintain high fidelity of the gene expressionprocess. In this review, focused primarily on mammalian TREX, we present the known compositionwith an emphasis on its dynamic organization uncovered over the last few years. We also summarizethe current knowledge regarding how it functions in gene expression. Finally, we will present newfields in which TREX seems to have an essential role.

Version of Record published:27 September 2016

Received: 22 January 2016Revised: 11 May 2016Accepted: 23 May 2016

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2911

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

The composition of TREXTREX consists of a stoichiometric salt-resistant hexameric core called THO, consisting of THOC1, 2, 5–7 andTex1 [10] together with a group of additional proteins. These can come together in different combinations tomake alternative forms of TREX [14–19] (Figure 2 and Table 1). The THO subunits were originally linked tomRNP biogenesis and export through their mutant phenotypes in S. cerevisiae [31,47,48]. Subsequently, theirexistence as a stable THO complex was discovered by their ability to suppress the transcriptional defects of theHpr1 mutant by overexpression [34]. The genetic and physical interaction of THO with the DEAD-box RNA

Figure 1. Overview of NXF1- and TREX-dependent mRNP formation and nucleocytoplasmic transport.

During gene expression, the TREX complex is recruited co-transcriptionally by a direct interaction with the phosphorylated C-terminal domain (CTD)

of the large subunit of RNA polymerase II and via the PRP19:U2AF2 complex. Each processing step undergone by the pre-mRNA (50-capping,co- and posttranscriptional splicing, 30-end processing) acts as a trigger for TREX assembly and deposition along the transcript. Once bound to the

mRNA, the combined action of adaptor (red and pink) and co-adaptor proteins (e.g. THOC5, CHTOP and CPSF6) recruit the NXF1:NXT1

heterodimer and cause a conformation change in NXF1, allowing exposure of its RBD for interaction with mRNA. In turn, the adaptor protein hands

the mRNA over to NXF1. This process is promoted by protein arginine methyltransferase 1 (PRMT1) through arginine methylation of both the

co-adaptor CHTOP, facilitating its interaction with NXF1, and the adaptor ALYREF, which reduces its RNA-binding activity enabling RNA handover

to NXF1. Additional adaptor proteins, such as SR proteins and ZC3H3, can also recruit NXF1 to mRNA. Subsequently, direct interactions of NXF1

with the TREX-2 complex and nucleoporins allow the mRNP to dock at the nuclear pore, thus promoting export of the mRNP. At the nuclear pore

export, adaptors are released from the mRNP, this is predicted to revert NXF1 to a low-affinity RNA-binding state that primes it for release from the

mRNP. DBP5 and GLE1 trigger the release of NXF1 from the mRNP on the cytoplasmic side of the nuclear pore for recycling back to the nucleus.

2912 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

helicase Sub2p and the RNA export adaptor Yra1p and equivalent biochemical interactions among the humanorthologs led to the discovery of the conserved TREX complex [6]. Additional mammalian TREX subunitshave been identified through molecular associations and functional characterization through depletion andoverexpression effects [10,14,17,24]. TREX subunits have discrete functions with diverse cellular roles, comingtogether to form this influential complex in the cell and the curriculum vitae for each subunit is presented below.

THOHuman THOC1 is the ortholog of the yeast protein Hpr1 [6,15] and was originally discovered by its associationwith the tumor suppressor retinoblastoma protein (pRB) [20]. THOC1 is involved in cell cycle regulation andinducing p53-independent apoptosis through its death domain (Figure 2) [49–52]. THOC2 is the humanortholog of the yeast Tho2 protein and, as the largest subunit of TREX, is proposed to act as a scaffold for theformation of the complex [53]. THOC2 is an essential component for maintaining TREX function in humans[15,42], and mutations in the THOC2 gene are associated with intellectual disabilities [22,54]. The conservedTHOC3 (hTEX1) protein has been recently established as a component of the THO subcomplex in TREX andcontains WD40 repeat motifs that allow multiple protein interactions (Figure 2) [10,15]. THOC5, 6 and 7 were

Figure 2. Schematics of the domain structures for mRNA export factors.

The major characterized domains within TREX subunits and NXF1 are shown. Each protein and domain is drawn approximately

to scale and the size of each protein in amino acids is shown on the right hand side. RBD, RNA-binding domain; R-rich,

arginine-rich; RS-rich, arginine/serine-rich; RRM, RNA recognition motif (not necessarily involved in RNA binding). UBM,

UAP56 (DDX39B)-binding motif; WxHD corresponds to the recently identified motif which binds EIF4AIII [12]. The other

domains are all well characterized. Note that THOC2 is not drawn to scale due to its size.

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2913

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

Table 1 Composition of the TREX complex

Humanname Alternative names Cellular function

Yeastorthologs kDa Functional features Key references

Link toBioGRID

THOsubunits THOC1 hHpr1, p84,p84N5, N5

Apoptosis regulator Hpr1p 76 Death domain, Rb-interactingfactor

[20] [21] [196] BIOGRIDENTRY

THOC2 hRlr1 THO scaffold protein Tho2p,Rlr1p

183 Coiled-coil domains,lysine-rich region

[31] [15] [22] BIOGRIDENTRY

THOC3 Tex1, hTrex45 Tex1p 39 WD40 domains [15] [10] BIOGRIDENTRY

THOC5 fSAP79, Fmip Export co-adaptor 79 Leucine zipper, Fms-bindingdomain

[23] [10] [24] BIOGRIDENTRY

THOC6 fSAP35, Wdr58 38 WD40 domain [10] [15] BIOGRIDENTRY

THOC7 fSAP24, Nif3l1bp1 24 Coiled-coil domain, Leucinezipper

[10] BIOGRIDENTRY

TREXsubunits DDX39B Uap56, Bat1, P47 Splicing factor,EJC-associated protein,RBP loading factor

Sub2p 49 ATP-dependent DEAD/DEAH-box RNA helicase

[25] [6] [26] BIOGRIDENTRY

DDX39A Ddx39, Urh49 Ddx39b paralogue,Putative EJC-associatedprotein

49 ATP-dependent DEAD/DEAH-box RNA helicase

[27] [28] BIOGRIDENTRY

ALYREF Aly, Ref, Bef, Thoc4 Export adaptor,EJC-associated protein

Yra1p,She11p

27 RRM, Nxf1-binding,Ddx39-binding, UBMsequence

[29] [6] [30] BIOGRIDENTRY

UIF Fyttd1 Export adaptor,EJC-associated protein

37 Functional homology withAlyref

[17] [186] BIOGRIDENTRY

LUZP4 Hom-Tes-85, CT-8 Export adaptor,EJC-associated protein

36 Functional homology withAlyref, RS-dipeptides,Leucine zipper

[33] [19] BIOGRIDENTRY

CHTOP Srag, C1orf77, Fop Export co-adaptor,EJC-associated protein

26 PRMT1-interaction domain,RGG box

[35] [36] [16] BIOGRIDENTRY

SARNP Cip29, Tho1, Hcc-1 Tho1p 24 SAP domain [37] [14] BIOGRIDENTRY

POLDIP3 Skar, KIAA1649,PDIP46EJC-associatedProtein

46 UBM-like sequence, RRM [38] [18] BIOGRIDENTRY

ZC3H11A Zc11a, ZC3HDC11A 89 Zinc finger protein [14] [18] BIOGRIDENTRY

ERH dDroer 12 Generally uncharacterized [197] [14] [39] BIOGRIDENTRY

Continued

2914©

2016The

Author(s).Thisisan

openaccess

articlepublished

byPortland

PressLim

itedon

behalfoftheBiochem

icalSocietyand

distributedunder

theCreative

Commons

AttributionLicense

4.0(CC

BY)

Biochem

icalJournal(2016)4732911

–2935DOI:10.1042/B

CJ20160010

Table 1 Continued

Humanname Alternative names Cellular function

Yeastorthologs kDa Functional features Key references

Link toBioGRID

TREX-associatedproteins

NXF1 Tap RNA export receptor Mex67p 70 Arginine-rich RBD,pseudo-RRM, NTF2-likedomain, UBA

[40] [41] [42] BIOGRIDENTRY

NXT1 p15 Required for Nxf1stabilization and mRNAexport

Mtr2p 15 Stabilizes and binds toNXF1’s NTF2-like domain

[198] [199] [43] BIOGRIDENTRY

ZC3H18 Nhn1 NEXT complexcomponent

106 CCCH-containing, Zinc-fingerprotein

[200] [44] [45] BIOGRIDENTRY

SRRT Ars2 pri-miRNAs processing,CBC effector in RNA 30

processing

100 Cap-binding protein [201] [202] [203] BIOGRIDENTRY

NCBP3 C17orf85, Elg Involved in RNA exportupon viral infection

71 Cap-binding protein, formingan alternative CBC withNcbp1

[204] [14] [46] BIOGRIDENTRY

NCBP1 Cbp80 Transcription elongation,RNA export, RNA stability

Cbc1p 71 Cap-binding protein, formingthe CBC with Ncbp2

[205] [11] [15] BIOGRIDENTRY

©2016

TheAuthor(s).This

isan

openaccess

articlepublished

byPortland

PressLim

itedon

behalfoftheBiochem

icalSocietyand

distributedunder

theCreative

Commons

AttributionLicense

4.0(CC

BY)2915

Biochem

icalJournal(2016)4732911

–2935DOI:10.1042/B

CJ20160010

originally identified as part of THO in human and Drosophila cells, but have no known yeast orthologs[7,10,24]. Mammalian THOC5 is a cytoplasmic substrate for the macrophage-colony stimulating factor receptortermed FMS [23] and is directly involved in the differentiation of macrophages [55], and a role in hematopoi-esis and cancer has been established [56]. The THO complex is implicated in embryonic stem (ES) cell self-renewal, being responsible for the export of pluripotency transcripts in mouse ES cells [57]. THOC7 is devoidof a nuclear localization sequence but forms a tight complex with THOC5, allowing the heterodimer to betranslocated to the nucleus [9]. THOC3 and THOC6 contain WD40 repeat motifs (Figure 2) commonly usedas protein interaction domains, which may contribute to THO assembly.

DDX39BDEAD-box protein 39B (DDX39B), widely known as U2AF65-associated protein 56 (UAP56), is the metazoanortholog of the yeast Sub2p protein. First discovered as an essential factor for pre-mRNA splicing through itsassociation with U2AF2 [25], this RNA-stimulated ATPase and DEAD-box RNA helicase promotes spliceo-some assembly [26,58]. DDX39B is present on pre-mRNAs during splicing [15,59] and is required for the sub-sequent recruitment of the TREX subunit ALYREF to both spliced and intronless mRNAs [59,60]. On bindingALYREF, the RNA bound to DDX39B is transferred to ALYREF [16]. This is a part of an orchestrated sequenceof RNA handover events that occur in nuclear mRNA export, whereby the mRNA acts like a baton in a relayrace. DDX39B accompanies the mRNP to the nuclear pore, but is eventually displaced before nucleocytoplas-mic translocation [61]. The displacement is triggered by recruitment of the mRNA export receptor NXF1,which binds to ALYREF in a mutually exclusive manner with DDX39B (Figure 1) [30]. In mammalian cells, aparalog of DDX39B exists, DDX39A, which has some functional redundancy with DDX39B, because depletionof both helicases is required to efficiently block mRNA export [17,27]. However, different groups of mRNAshave their export affected following knockdown of DDX39A or B, indicating that the two helicases most likelyassemble into subtly different TREX complexes acting preferentially on specific groups of mRNAs [28].

ALYREFALYREF was originally described as a transcriptional coactivator of the T-cell receptor α gene by bindingLEF-1 and acute myeloid leukemia (AML)-1 transcription factors to form an enhancer-stimulating complex[29]. It was subsequently characterized as a molecular chaperone capable of stimulating the dimerization ofbasic leucine-zipper transcription factors, thereby inducing their DNA-binding activity. These studies alsoshowed that ALYREF has a propensity to form an oligomeric complex in vitro with a molecular weight of460 kDa corresponding to 14 ALYREF monomers [62]. ALYREF contains N- and C-terminal transient helices,known as the UAP56-binding motif (UBM), which are necessary and sufficient for interaction with DDX39A/B[17] (Figure 2). It also has a central RNA recognition motif (RRM), which binds RNA weakly through loops 1and 4 [63]. The RRM is flanked by two unstructured arginine-rich regions that are the principal RNA-bindingsites. Such separate RNA-binding domains set within unstructured flexible peptides [63] would provideALYREF with the potential to bridge interactions between separate parts of an mRNA, which is consistent withthe RNA annealing activity observed for its yeast ortholog, Yra1 [64]. Such an activity suggests that ALYREFmay play an important role in packaging of the mRNP, which adopts a compact crescent-shaped structureduring translocation from the site of synthesis to the nuclear pore [65]. The arginine-rich regions are also theprincipal sites for interaction with the mRNA export receptor NXF1, and binding of NXF1 to ALYREF triggerstransfer of RNA from ALYREF to NXF1 [30]. Arginines within the RNA-binding region of ALYREF aremethylated. These modifications have been shown to reduce the ability of ALYREF to bind RNA which isimportant for the handover of RNA from ALYREF to NXF1 [66]. ALYREF binds phosphoinositides in vitroand mutations in the N- and C-terminal arginine-rich regions alter this binding. However, whether this associ-ation is physiologically important remains unclear since the mutations that alter phosphoinositide binding,leading to altered ALYREF localization in vivo, may also have altered its RNA-binding activity [67]. LikeDDX39A/B, ALYREF is displaced from the mRNP before nucleocytoplasmic translocation [61,68]. ALYREF isinvolved in the export of both spliced and intronless mRNAs [69], and RNA interference (RNAi)-mediatedknockdown of ALYREF leads to disruption of mRNA export for around 3900 mRNAs in 293T cells [70].Functional redundancy between ALYREF and other mRNA export factors such as UIF (described below) mayaccount for the relatively small number of mRNAs whose export is affected following ALYREF RNAi.

2916 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

UIFUIF (also known as FYTTD1) was first identified using a BLAST search with the ALYREF UBM and, althoughit harbors this motif, it has relatively little additional homology with ALYREF and other mammalian proteinsoutside this peptide. UIF functions in a redundant manner with ALYREF and knockdown of both proteinsleads to a strong mRNA export block in mammalian cells. Knockdown of ALYREF also leads to a largeup-regulation of UIF levels in cells, which probably accounts for the modest block in mRNA export seen fol-lowing ALYREF RNAi [17]. Although UIF associates with ALYREF, this interaction is RNA-dependent,whereas the association with other TREX subunits is not [17]. Thus, UIF may form an alternative TREXcomplex without ALYREF and the RNA-dependent association with ALYREF may arise because multipleTREX complexes are loaded onto an mRNA.

LUZP4LUZP4 was originally characterized as a cancer/testis antigen [33], which represents a group of poorly charac-terized proteins whose expression is normally restricted to testis but are frequently up-regulated in cancer cells.Subsequently, a BLAST search revealed that LUZP4 also carries a UBM (Figure 2) and acts as an mRNAexport factor associating with TREX subunits and complementing ALYREF RNAi in vivo. While the expressionof LUZP4 is normally restricted to testis, it is up-regulated in melanoma cancer cells where it is required fortheir survival [19].

CHTOPCHTOP was originally identified as an RNA-binding protein that regulates the cell cycle [71]. It was subse-quently shown to be a binding partner for protein arginine methyltransferase 1. It is involved in the expressionof the γ-globin gene and is recruited to the estrogen target gene, pS2, in breast cancer cells [35,72]. Its role ingene expression is further shown by its presence in the ‘five friends of methylated CHTOP’ desumoylationcomplex that is recruited to the Zbp-89 transcription factor during transcriptional activation [36]. The directfunction of CHTOP in transcriptional regulation is akin to that of ALYREF. Therefore, CHTOP may also bekey in linking the stages of transcription, pre-mRNA processing and export as a component of TREX. CHTOPcontains two copies of the UBM found in ALYREF, UIF and LUZP4 (Figure 2) and is capable of stimulatingthe ATPase and helicase activities of DDX39A/B. Both ALYREF and CHTOP are dependent on DDX39A/B forefficient loading onto mRNA in vivo [16].

SARNPSARNP (also known as CIP29) was first identified as an EPO-stimulated cytokine-induced protein involved incell cycle progression [37]. The small protein contains a SAP DNA-binding motif (Figure 2), suggesting a pos-sible role in direct transcriptional regulation and it was subsequently identified as a TREX component [14]. Itsyeast ortholog, Tho1, was identified alongside Tho2 as suppressing transcriptional defects of hpr1 mutants [31],forming the initial connection to TREX [31,73]. SARNP forms an ATP-dependent trimeric complex withDDX39A/B and ALYREF [14]. Moreover, SARNP stimulates the ATPase and helicase activities of DDX39A/B[16,74]. The plant ortholog of SARNP is known as MOS11 where it has also been shown to facilitate mRNAexport [75].

POLDIP3POLDIP3 was identified as an S6 kinase substrate with homology to ALYREF, which contributes to cell growthregulation [38]. It localizes to nuclear speckles, but also enhances translation [76], so is likely to play a role inmany stages of mRNP biogenesis. It associates with DDX39A/B and TREX in an ATP-dependent manner andits over expression leads to retention of poly(A)+ RNAs in nuclear speckles [18].

ZC3H11AZC3H11A co-purifies with multiple other TREX subunits [14] and recent work indicates that, similar toPOLDIP3, it associates with TREX and DDX39A/B in an ATP-dependent manner. Knockdown of ZC3H11Aby RNAi leads to a strong nuclear accumulation of mRNA [18]. Together, these data suggest that ZC3H11A isa bone fide TREX subunit, required for efficient mRNA export.

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2917

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

Other proteins associated with TREXUsing immunoprecipitation with antibodies to THOC2, SARNP and DDX39A/B, a group of core proteins thatassociate with all three TREX subunits were identified using mass spectrometry. These included CBP80,CBP20, SRRT and NCBP3 (ELG), which all associate with the 50 cap on mRNA and ERH [14]. ERH has mul-tiple roles in the cell including roles in splicing and the cell cycle (reviewed in ref. [39]) and is a bindingpartner for POLDIP3 [77] and this may account for its association with TREX.

Differences in TREX between speciesThe two major systems which have been used to study TREX are yeast and mammalian. While TREX is clearlyconserved between these organisms (Table 1), there are also some important differences. For example, there areno known yeast orthologs for CHTOP, POLDIP3 and ZC3H11A. There is also divergence among the THOsubunits. THOC1, 2 and 3 have orthologs in the two systems, but THOC5, 6 and 7 are specific to higher eukar-yotes [7], with the yeast THO complex having two alternative subunits, Mtf2 and Thp2 [6]. Humans have asingle ALYREF gene, whereas mice and yeast have two orthologous genes. UIF is conserved in mammals, butis absent from yeast and other higher eukaryotes such as Drosophila and Caenorhabditis elegans. LUZP4 is con-served in vertebrates but absent in other metazoans. These important species differences, with an expansion ofTREX subunits during evolution, probably reflect the diverse biological roles that TREX plays particularly inmulticellular organisms.

Recruitment of TREX to mRNA through transcription andRNA processingTranscriptionAs a dynamic complex, TREX components are recruited to the developing mRNP at various stages of biogen-esis. Chromatin remodeling and subsequent transcription form the first phase of mRNA generation. UIF isrecruited to the transcription complex and the nascent RNA by association with the SSRP1 subunit of FACT[17], a histone chaperone and transcription elongation factor that remodels the H2A:H2B histone dimer, soRNA Pol II can transcribe the gene [78]. ALYREF is recruited by another histone chaperone Spt6, which worksalongside FACT to remodel H3:H4 associated nucleosomes during transcription [79]. Spt6 binds the carboxy-terminal domain (CTD) of the large subunit of RNA Pol II and recruits IWS1. IWS1 in turn recruits ALYREFto actively transcribing genes [80].The yeast TREX complex associates directly with the Pol II CTD; however, the interaction is partially

dependent on the presence of nascent RNA [81,82]. This mechanism may ensure that TREX only associateswith transcribing Pol II. Ser2 phosphorylation of the Pol II CTD is required for TREX binding and its occu-pancy on genes mirrors Ser2 phosphorylation, increasing in a 50 to 30 direction. When the increased recruit-ment of TREX towards the 30-end of a gene is impaired, this specifically hinders the expression of long genes[81]. As TREX assembles on nascent RNA, there is a possibility that the CTD could become depleted of TREXsubunits during transcription and this would be particularly acute on long genes. Therefore, Ser2-dependentenhanced recruitment of TREX to the CTD may ensure that it does not become depleted of TREX subunitsduring transcription of long genes. Interestingly, Mex67, the yeast ortholog of NXF1, is also necessary for Hpr1(THOC1) stabilization during transcription [83], demonstrating the feedback and coordination between tran-scription and export steps.The Prp19 complex, which functions in splicing, transcription and transcription-coupled DNA damage

repair [84], is also required for efficient recruitment of TREX to transcribed genes in yeast. A mutation in theSyf1 subunit of the Prp19 complex results in less Hpr1, Sub2 and Yra1 associated with genes, especially at the30-end, suggesting that Prp19 acts to recruit TREX co-transcriptionally [85]. The human PRP19 complexassociates with U2AF2 and together they promote Pol II CTD-dependent splicing activation [86]. Moreover,the PRP19 complex associates with TREX in human cells [14]. U2AF2, which directly binds DDX39A/B, mayprovide the link between TREX and the PRP19 complex, facilitating co-transcriptional recruitment of TREX totranscribed genes. An increasingly recurrent theme in gene expression is the coupling of each stage and recipro-cal relationships between each step. It appears that this is also the case for transcription and export becauseTREX is required for efficient transcription elongation, particularly of long genes [21,87]. ALYREF affects tran-scription of a subset of genes [70], suggesting a reciprocal coupling between transcription and export viaALYREF, as it has been observed for other steps in the gene expression pathway [88].

2918 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

50 Cap formationThe NCBP1 (CBP80) and NCBP2 (CBP20) heterodimer forms the CBC, which associates with the 50 cap.TREX is recruited to the 50 cap by direct NCBP1–ALYREF and NCBP1–THO interactions [11,15].Interestingly, SRRT also co-purifies with TREX [14]. SRRT associates with the CBC, the nuclear exosome tar-geting complex (NEXT) and together with ZC3H18 forms the CBC–NEXT complex [45]. ZC3H18 has inde-pendently been shown to recruit TREX to the hepatitis B virus post-transcriptional regulatory element andpromote export of intronless mRNAs [44]. NEXT consists of three subunits, hMTR4, RBM7 and ZCCHC8,and multiple TREX subunits (ALYREF, CHTOP, THOC1,2,3,5,7, POLDIP3, UIF and DDX39A) co-purify withhMTR4 [45]. This raises the possibility that TREX bound to MTR4 might regulate the assembly of CBC–NEXT and the recruitment of the exosome. In this way, the assembly of TREX on the 50 cap may be a criticaldecision point regarding whether an mRNA is degraded or exported during nuclear quality control. NCBP3has recently been characterized as an alternative binding partner for NCBP1 at the cap, which can replaceNCBP2. NCBP3 knockdown leads to a clear nuclear accumulation of mRNA, establishing its role in mRNAexport. It also appears to act preferentially during times of stress such as during viral infection [46]. The earlierobservation that NCBP3 associates with TREX [14] may account for the role of NCBP3 in mRNA export, byproviding the means to ensure TREX recruitment to the 50 end of mRNAs and promoting efficient mRNAexport in times of stress. The length of an RNA determines its export pathway and RNAs shorter than 300bases generally do not utilize the TREX:NXF1 pathway. This choice is determined early during transcriptionwhen hnRNP C associates with the 50-end of the nascent RNA if it is long enough. hnRNP C binding to the50-end of the nascent RNA prevents recruitment of alternative export factors such as PHAX. This directs theRNA to the mRNA export pathway, whereby TREX probably displaces hnRNP C from the cap proximal region[89].

SplicingIt has long been known that the presence on an intron enhances gene expression from an otherwise intronlesscDNA and that splicing enhances mRNA export [90]. Using in vitro splicing reactions, it was shown thatrecruitment of human TREX is splicing-dependent [10,59]. The splicing dependence may be linked to the factthat DDX39A/B is involved in spliceosome assembly and additionally binds U2AF2, which recognizes the poly-pyrimidine tract within introns, early during spliceosome assembly. U2AF2 also co-operates with DDX39A/Bto regulate nuclear retention of pre-mRNA [91]. Therefore, rearrangements of the U2AF2:DDX39A/B axisduring splicing might couple TREX assembly on spliced mRNA with release of pre-mRNA retention signals.Interestingly, intron status is significant in yeast, despite direct co-transcriptional recruitment, as Yra1 isrecruited preferentially to spliced mRNA [92]The ability of TREX to be loaded onto spliced mRNA in vitro occurs in the absence of transcription and

30-end cleavage/polyadenylation [10]. Furthermore, approximately 80% of splicing occurs co-transcriptionallyin human cells [2], and so a considerable amount of TREX may be loaded onto mRNAs prior to 30-end pro-cessing. Co-transcriptional splicing is thought to occur largely in regions of decompacted chromatin at the per-iphery of, or within, nuclear speckles. Moreover, TREX subunits interact in vivo on the periphery of nuclearspeckles [93], suggesting that this is a major site for co-transcriptional TREX assembly. In contrast, post-transcriptional splicing appears to be largely restricted to nuclear speckles [2] and post-splicing release ofmRNAs from speckles is dependent on TREX subunits [94].Following splicing, the EJC is loaded onto the mRNA at a canonical position 24 bases upstream of the splice

junction [95], furthermore the EJC co-purifies with TREX subunits [96,97]. These data led to the suggestionthat the EJC provides a binding platform for mRNA export factors including the TREX subunit ALYREF andthe mRNA export receptor NXF1 [96]. More recently, a specific sequence motif has been identified withinALYREF (WxHD) (Figure 2) that facilitates its interaction with the EJC subunit EIF4AIII. Thus, the EJC andCBC provide a stable binding platform for TREX on the 50-end of mRNAs in vitro [12]. The involvement ofthe EJC in TREX loading is consistent with the recent observation that the EJC assembles on mRNAs inregions surrounding the nuclear speckles, termed perispeckles [98] (Figure 1), where TREX subunits also inter-act [93]. Whether mammalian TREX is loaded onto internal mRNA sites around EJCs in vivo remains to beseen. In yeast, this appears to be the case, with TREX subunits bound along the body of mRNAs [92,99].Despite the central importance of splicing for normal mRNA export, when splicing is inhibited by drugs

such as spliceostatin, this leads to the leakage of pre-mRNA to the cytoplasm [100]. However, the overall

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2919

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

amount exported is very small and the vast majority of unspliced pre-mRNA is retained in the nucleus whenobserved by fluorescence in situ hybridization [101]. Nevertheless, since some pre-mRNA does leak to the cyto-plasm, this may indicate that export factors can be loaded onto unspliced pre-mRNA in vivo. Thus, one of thekey effects of splicing in mRNA export may be to ensure nuclear retention of pre-mRNA already loaded withexport factors.While intron removal is important for the export of spliced mRNAs, TREX subunits also associate with

intronless mRNAs [69] and the association between CBC and TREX is important for their export [102].Interestingly, U2AF2 also promotes intronless mRNA export in Drosophila [103], which may be connectedwith its ability to bind DDX39B. Specific internal RNA elements have been identified which promote intronlessmRNA stability and export [104,105]. These elements bind U2AF2, PRP19 complex and TREX subunits and,therefore, may act as surrogates for splicing on intronless mRNAs. The recruitment of TREX to internal siteson mRNAs may be important to ensure the binding of multiple copies of the export receptor NXF1, ensuringsmooth passage of the elongated mRNP through the nuclear pore. While TREX can function as a binding plat-form for NXF1, some intronless mRNAs appear to utilize additional strategies to ensure NXF1 recruitment.For example, histone mRNAs are bound by SR proteins that can in turn directly recruit NXF1 in a mannersimilar to ALYREF [106–108].

30-end processingA necessary pre-requisite for mRNA export from the nucleus to the cytoplasm is the release of mRNA fromthe DNA template, which is coupled to pre-mRNA 30-end processing [3]. In yeast, two key proteins, Pcf11 andClp1 come together to cause pre-mRNA cleavage. Pcf11 is recruited early during transcription through the PolII CTD and also recruits Yra1 at the 30-end of genes. Clp1 recruitment to Pcf11 triggers displacement of Yra1from Pcf11 [13]. The ability of Yra1 to regulate accessibility of Clp1 to Pcf11 means that it can influence thesite of cleavage/polyadenylation within a pre-mRNA, particularly at genes with divergent efficiency elements[109]. The displacement of Yra1 from Pcf11 by Clp1 is enhanced by the presence of Sub2, suggesting that theprocess is coordinated with loading Yra1 onto mRNA. Intriguingly, in both tho and sub2 mutants, a stalledintermediate in mRNP biogenesis is created in which nuclear pore components and polyadenylation factorsremain associated with chromatin in what is known as a ‘heavy chromatin’ fraction [110]. Together, these dataindicate that TREX plays a key role in 30-end processing and the concomitant release of the mRNP fromchromatin.The PCF11-ALYREF interaction is conserved in humans [13] and DDX39B has been found to be present in

a highly purified 30-end processing complex [111]. Thus, poly(A) site choice in mammalian cells is also likelyto be governed by ALYREF, CLP1, DDX39A/B and PCF11, through similar mechanisms to those used in yeast.Additionally, THOC5 binds two 30-end processing factors, CPSF100 [112] and CPSF6 [113]. Through theseinteractions, THOC5 can regulate poly(A) site choice for specific genes. In the case of CPSF6, THOC5 directsits early recruitment to transcribed genes and, following THOC5 or CPSF6 loss, proximal poly(A) sites arepreferentially used [113]. A further twist in the ‘tail’ arises from recent studies on CDK11 that triggers Ser2phosphorylation of the Pol II CTD [114]. Ser2 phosphorylation peaks at the 30-end of genes and this stimulatesthe recruitment of 30-end processing factors to the gene. TREX forms a stable complex with CDK11 and isrequired for its recruitment to the 30-end of HIV 1 genes and subsequent CTD Ser2 phosphorylation.Therefore, TREX orchestrates 30-end processing via CDK11 on HIV transcripts, but the extent to whichTREX works with CDK11 on cellular mRNAs remains to be seen. In summary, TREX acts at many points inthe evolution of the 30-end of mRNA through interactions with multiple components of the transcriptionand 30-end processing machinery. In turn, 30-end processing, a necessary pre-requisite for mRNA export, nodoubt allows the evolution of TREX to a state where it can subsequently trigger export of the fully processedmRNA.

Integrating mRNA processing with TREX assemblyThe early assembly of TREX subunits probably occurs on the RNA Pol II CTD through multiple mechanisms,including direct binding of subunits to the Ser2 phosphorylated CTD and indirectly via the PRP19:U2AF2complex. Subsequently, TREX subunits transfer to the nascent mRNP. This process is driven in part by theRNA helicases DDX39A and B, which load export factors such as UIF, ALYREF, LUZP4 and CHTOP onto themRNP, using the helicase ATPase cycle [14,16] (Figure 3). The assembly on the mRNP is driven by the majorpre-mRNA processing events (capping, splicing and polyadenylation) that provide their own protein signatures

2920 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

such as the CBC and EJC. These protein signatures probably serve to stabilize TREX at multiple positionsalong the mRNP (Figure 1). Interestingly, subunits of the THO complex are largely devoid of RNA-bindingactivity in yeast with the exception of Tho2 [53], and in mammals, THOC5 binds RNA weakly [42]. In contrastDDX39A/B, ALYREF and CHTOP bind RNA avidly [16] and these may well provide the main connectionbetween TREX and the mRNA. Whether all ALYREF and CHTOP molecules associated with the mRNP arealso bound by THO is not clear. Analysis of the relative amounts of mRNA export and RNA processing factorsin mouse cells shows that DDX39B, SARNP, ALYREF and CHTOP are present in considerable excess overTHO subunits and the RNA Pol II large subunit (Figure 4). For example, there is 90 times more DDX39B thanTHOC5 in the cell [115]. While ALYREF, CHTOP, SARNP and DDX39B are all present in excess of the cap-binding protein NCBP1, THO subunits are present in substoichiometric amounts, with the most abundantTHO subunit, THOC7 being present at a ratio of approximately 1:2 with NCBP1. These variations in the levelsof TREX subunits in the cell may reflect differential recruitment of specific TREX factors to subsets of mRNAs.In favor of this hypothesis, depletion of THOC5 alters the export of approximately 2.9% of mRNAs in mousecells and depletion of multiple THO subunits in Drosophila only effects export of approximately 20% of thetranscriptome [7]. However, the restricted sets of mRNAs affected could also be caused by functional redun-dancy between export factors such as that seen between ALYREF and UIF [17]. THO may alternatively play itsmajor role in export by chaperoning the recruitment of ALYREF, DDX39B, SARNP and CHTOP onto mRNAthrough its association with RNA polymerase II. Consistent with this model, THO levels are much more com-parable with RNA Pol II large subunit levels (Figure 4) and in yeast, chromatin immunoprecipitation experi-ments reveal that THO associates with the majority of transcribed genes [116]. The two possibilities are notmutually exclusive, but what is clear is that there are likely to be subtly different forms of TREX associating ondifferent mRNAs and within a single mRNP, there are likely to be varying flavors of TREX present. The >3fold excess of DDX39B, ALYREF and SARNP, which form a stable ATP-dependent trimer [14] over thenuclear cap-binding proteins (Figure 4), indicate that the cell has the opportunity to recruit multiple copies ofthis trimer to a single mRNP. Thus, these proteins may play an important role in mRNP packaging as well asexport, consistent with the RNA annealing activity of Yra1 (ALYREF) [64]. Finally, it is striking that PCF11 issuch a low abundance protein relative to THO or other mRNA export factors, with approximately 1000 timesmore ALYREF present in the cell. This vast excess of ALYREF probably helps ensure that PCF11 remains satu-rated with ALYREF until 30-end processing, thus guarding against inappropriate poly(A) site choice. The sub-stoichiometric amounts of PCF11 with respect to RNA polymerase II may indicate that it is used on selectivetranscripts in mammalian cells.

Biological roles of TREXmRNA exportTREX plays a central role in mRNA export and this activity is governed by its ability to act as a binding platformfor NXF1 [42]. Nuclear export factor 1 (NXF1, also called tip-associated protein, TAP) was originally identified ina yeast two-hybrid screen to identify cellular partners of tyrosine kinase-interacting protein (Tip), a viral proteinfrom Herpesvirus saimiri [117]. A link between NXF1 and mRNA export was revealed by a synergistic combin-ation of yeast genetic studies and biochemical experiments performed in metazoan systems. The yeast proteinMex67p was first shown to be essential for bulk poly(A)+ RNA nuclear export, consistent with its nuclear localiza-tion, its ability to bind mRNAs in vivo and to contact the nuclear pore complex [118]. Concurrently, NXF1 wasshown to promote nuclear export of retroviral transcripts by binding to RNA secondary structures called constitu-tive transport elements (CTEs) [119]. Subsequently, it was shown that Mex67p’s function in mRNA export is con-served in metazoans through its ortholog NXF1 [40].NXF1 is a multidomain protein (Figure 2), with each domain connected by flexible linkers. Extensive struc-

tural characterization has revealed an unstructured N-terminal region preceding the following folded domains:an RRM fold, a leucine-rich repeat, an NTF2-like (NTF2L) domain and finally a C-terminal UBA domain[43,120,121]. The NTF2L domain forms a tight complex with NXT1, which is essential for the stability andactivity of NXF1 [43]. The NTF2L and UBA domains provide separate binding sites for FG repeat sequencesfound in the nucleoporins lining the nuclear pore. These FG repeat binding sites help direct NXF1 to thenuclear pore [43].An additional conserved protein complex known as TREX-2, which consists of GANP, PCID2, DSS1 and ENY2

in mammals, is involved in the docking of NXF1 at the nuclear pore during mRNA export (Figure 1, [122,123]).

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2921

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

Figure 3. The life cycle of the TREX complex.

The THO complex constitutes a salt-resistant core of tightly associated proteins (light blue). During gene expression, it dynamically associates

with a variety of proteins (red, orange, yellow, purple and gray) to form the TREX complex, whose composition evolves during mRNP

formation. THO is able to recruit the adaptor proteins (red), the co-adaptor CHTOP and the RNA helicase DDX39B. The DDX39B RNA helicase

is thought to use rounds of ATP hydrolysis to load adaptors and co-adaptors onto the RNA. In turn, they recruit the mRNA export receptor

NXF1:p15, which displaces DDX39B. While the co-adaptor THOC5, as a part of THO, is likely to associate early with the NTF2L domain of

NXF1, arginine methylation of the second major co-adaptor CHTOP is a prerequisite for it to bind that same domain. This suggests that

rearrangements occur within TREX. It is currently unclear whether the additional subunits (gray) are all part of the same TREX complex or

belong to variants of a remodeled TREX complex. It is also unknown whether the same THO complex is recycled for further rounds of mRNP

assembly (putative step 4).

2922 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

Certain subunits of TREX-2 are stably associated with the nuclear pore, but whether this association requireson-going transcription is currently unclear since conflicting data exist in the literature [122,124]. Work on theyeast complex has shown that it is involved in the translocation of genes to the nuclear pore and provides adirect connection between a gene’s promoter, transcription and the nuclear pore through the mediatorcomplex [125]. Furthermore, the ENY2 subunit of TREX-2 is also a component of the SAGA transcriptionactivation complex [126], and thus TREX-2 may use multiple connections with the transcription machineryto direct a gene to the nuclear pore. Studies in the human system have shown that GANP is involved inthe selective export of specific mRNAs [127,128], particularly those involved in gene expression steps,including RNA processing, splicing and ribosome biogenesis. GANP uses its FG repeat-like domain tointeract with the FG repeat-binding regions of NXF1 and stimulates a local high concentration of NXF1 at thenuclear pore [122]. Therefore, TREX-2 may provide a fast track for the export of certain transcripts bypositioning the gene close to the nuclear pore and promoting loading of NXF1 onto the mRNP and subsequentexport. The use of TREX-2 by certain genes may be important for cells to respond rapidly to cellularstresses [128].A systematic study of the RNA-binding activity of NXF1 domains using UV cross-linking revealed that the

major RNA-binding activity resides within the N-terminal unstructured region amino acids 61–118, withweaker RNA-binding activity identified within amino acids 1–60 and the RRM (amino acids 119–198) [30].These studies are corroborated by in vivo mRNP capture assays, which mapped the mRNP-binding site forNXF1 to amino acids 61–140 [129]. The presence of an RRM has caused some confusion in the literature overthe years regarding the NXF1 mRNA-binding activity. In fact, the major RNA-binding domain (RBD) corre-sponds to amino acids 1–118, not the RRM (amino acids 118–198). Moreover, the LRR, NTF2L and UBAdomains have no RNA-binding activity when assayed by UV cross-linking [30], though weak RNA-binding

Figure 4. The number of protein molecules per cell for mRNA export and processing factors.

This graph shows the average number of protein molecules per cell for a mouse NIH3T3 fibroblast cell line. The data were extracted from ref. [115].

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2923

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

activity has been detected for the NTF2L domain using electrophoretic mobility shift assays [130]. The NXF1RBD is rich in arginines and mutation of 10 of these arginines prevents NXF1 mRNA binding both in vitroand in vivo. Moreover, a mutant form of NXF1, which carries the 10 arginines mutated to alanine, fails to func-tion in mRNA export in vivo [30], establishing the RBD as the major determinant for mRNA binding. Thebinding of NXF1 to retroviral CTE elements appears to be fundamentally different from binding to mRNA andinvolves extensive interactions between the RRM, LRR and NTF2L domains of NXF1 [41,121,130].Interestingly, the NXF1 gene harbors a CTE-like structure within intron 10 and NXF1 binding to this sequencepromotes the export of a retained intron transcript that encodes a short form of NXF1, though the function ofthis short form of NXF1 is unclear [131].ALYREF binds to the N-terminal region of NXF1, including the RBD and RRM domains [132] (Figure 2). As

ALYREF binds mRNA avidly, it was given the name mRNA export adaptor, because it was thought to bridge theinteraction between RNA and NXF1. However, subsequent studies showed that when NXF1 binds to an ALYREF:RNAcomplex, the RNA is handed over to NXF1 [30] and ALYREF no longer binds RNA. This is consistent withbiochemical studies, showing that the NXF1-binding site and RNA-binding sites on ALYREF overlap [63].Additional mRNA export adaptors have been identified including various SR proteins, which, like ALYREF, usean arginine-rich peptide to bind the RBD + RRM domains of NXF1 [106–108]. A common feature of the RNAhandover process from an export adaptor to NXF1 is that once the adaptor protein is bound to NXF1 itenhances NXF1 RNA binding activity [30]. The reasons for this were unclear until it was realized that NXF1forms an intramolecular interaction between the RBD and the NTF2L domains which suppresses RNA binding(Figure 3) [42]. Thus, the NTF2L domain of NXF1 is autoinhibitory for mRNA binding driven by the RBD.ALYREF is involved in disrupting the NXF1 intramolecular interaction, which exposes the RBD for interactionwith RNA. A second site for interaction between TREX and NXF1 involves the NXF1 UBA domain that bindsTHOC1 [42]. The third site is the NTF2L domain, which THOC5 binds on the opposite side to that bound byNXT1. The binding of ALYREF to NXF1 stimulates the binding of THOC5 to the complex; on the basis of thisactivity, THOC5 was named a co-adaptor for mRNA export [24]. The NXF1 RBD binds to the NTF2L domainat a site overlapping that bound by THOC5 and it is the combined action of ALYREF and THOC5 binding toNXF1 that exposes its RBD, allowing optimal RNA-binding activity [42]. Since NXF1 requires both an adaptorand a co-adaptor for optimal RNA binding, this raises the question of whether SR proteins function independ-ently to recruit NXF1 to mRNAs, given they would only provide the adaptor function. Evidence from yeastsuggests that SR proteins may function in collaboration with TREX during mRNA export [133–135], and thismay be to ensure that a co-adaptor is available at such sites for optimal NXF1 RNA binding.THOC5 is not the only co-adaptor protein that binds the NTF2L domain of NXF1; CHTOP, RBM15 and

CPSF6 also bind this domain [16,136,137]. Similar to THOC5, CHTOP binding to NXF1 is stimulated by thepresence of ALYREF [16]. CHTOP and THOC5 are found in the same TREX complex bound to NXF1; there-fore, there is potentially sequential binding of these factors to NXF1 during mRNP maturation (Figure 3). Themethylation of arginines within CHTOP is essential for its interaction with NXF1 [16] and methylation of argi-nines within ALYREF is required for RNA handover to NXF1 [66]. Therefore, arginine methylation plays avital role in the activity of TREX during mRNA export.An interesting feature of two of the known NXF1 co-adaptors, THOC5 and CPSF6, is their involvement in

30-end processing and polyadenylation. Furthermore, NXF1 loss in cells leads to hyperadenylation of mRNAand loss of Mex67 in yeast leads to retention of the 30-end processing factors Rna14 and Rna15 on the mRNA[138]. A further factor involved in the recruitment of NXF1 to the mRNP is ZC3H3 and loss of this factorleads to mRNA hyperadenylation, similar to that seen following NXF1 depletion [139]. These observationssuggest that an adaptor, such as ALYREF, delivered via PCF11 and a co-adaptor, such as THOC5, deliveredthrough interactions with 30-end processing factors provide a key signal at the 30-end of mRNAs. This signal isinterpreted by NXF1 that subsequently binds to TREX, allowing correct evolution of the 30-end processingcomplex to a state suitable for mRNA export.Once NXF1 has been delivered to the mRNP by TREX, the mRNP is escorted to the nuclear pore [128].

Studies on Chironomus tentans have shown that ALYREF and DDX39B dissociate from the mRNP on thenuclear side or during translocation through the nuclear pore [61]. This is predicted to revert NXF1 to a low-affinity RNA-binding state and this may be an important prerequisite for its subsequent removal from themRNP. Dbp5, alongside its ATPase activator Gle1 and inositol hexakisphosphate phosphate 6, work togetherwith NUP159 to remodel the mRNP on the cytoplasmic side of the nuclear pore, triggering removal of NXF1[140–142]. NXF1 is then reimported into the nucleus for subsequent rounds of mRNA export.

2924 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

piRNA biogenesisWhile the majority of work on TREX has thus far focused on its role in mRNA export, recent studies indicatethat it has additional roles in piRNA biogenesis. piRNAs are short RNA molecules produced in germ line cellswith a major function in suppressing transposons during development. The first indication that TREX may beinvolved in piRNA biogenesis came from the observation that DDX39A/B co-localized at piRNA clusters withRhino, which is required for transposon silencing [143]. Moreover, DDX39A/B mutations disrupted a cytoplas-mic structure known as the nuage, which is rich in components of the piRNA processing machinery. Mostrecently, it has been shown that additional TREX subunits are involved in piRNA transcription and biogenesis,with TREX loading onto piRNAs being driven by a chromatin-associated protein, Cutoff [144]. However, it isnot clear yet whether TREX plays a role in the transport of piRNA precursors from the nucleus to thecytoplasm.

Genome stability, R-loops and cancerDuring transcription, the nascent RNA can hybridize with the single-stranded template DNA present withinthe transcription bubble. This structure is known as an R-loop and it leads to the opposing DNA strand beingleft single-stranded, making it susceptible to cleavage which can eventually lead to various forms of DNAdamage in cells [145,146]. TREX is intimately associated with both the RNA Pol II CTD and nascent RNA,leaving it poised to play a major role in sequestering the nascent RNA and preventing it from hybridizing tothe template DNA strand. A large increase in R-loops is found in TREX-depleted yeast and human cellsleading to far more DNA damage, transcription-associated recombination and DNA replication obstacles[87,116,147,148]. R-loops also form part of a natural process of genomic recombination to generate variation,such as for antibody class-switching in B-cells. THO depletion in murine cells results in an increased rate ofclass-switching as more R-loops are formed and consequently, a greater rate of recombination can occur [87].An RNA/DNA helicase, SETX (senataxin; Sen1 in yeast), is required to resolve R-loop formation. Interestinglyin yeast, Sen1 co-purifies with Yra1 (ALYREF) [149], raising the possibility that as well as packaging RNA tohelp prevent R-loop formation, ALYREF might also be involved in the regulation of SETX activity. A furtherrole for TREX in maintaining genome stability arises from studies on IPMK, which is involved in inositol phos-phate production in human cells. Loss of this kinase prevents the export of mRNAs encoding proteins involvedin the DNA damage response. ALYREF is implicated in this process and thus, TREX maintains appropriatelevels of proteins required to maintain genome stability by ensuring efficient export of their mRNAs [150].Since TREX can associate with various different adaptor proteins such as UIF and LUZP4 (Figure 3), thisraises the possibility that alternative forms of TREX promote the export of specific classes of mRNA [128].Given the importance of TREX in maintaining genome stability, it is unsurprising that TREX has been impli-

cated in many forms of cancer. CHTOP is recruited to the pS2 promoter, inducing the expression of estrogentarget genes in breast cancer cells [35,36] and thereby maintaining growth of the tumor. CHTOP is also requiredfor the expression of key genes required for the maintenance of tumor cells in glioblastoma [151]. THOC1 associ-ates with the tumor suppressor pRB, resulting in the prevention of cell cycle arrest and THOC1-induced apop-tosis [20,49,50]. This links THOC1 with tumor survival as mRNP biogenesis cannot occur efficiently enough tomaintain rapid cell division in its absence [152,153]. The up-regulation of THOC1 and THO complex expression,readily seen in a range of cancer types, may correlate with tumor size and have a greater effect in hormone-dysregulated cancers [152–155]. Phosphorylation of THOC5 on tyrosine 225 promotes its incorporation intomRNPs and THOC5 phosphorylation by leukemogenic protein tyrosine kinases is increased in patients withchronic myeloid leukemia [156]. ALYREF also shows an altered expression pattern in various cancerous tissues,but is more frequently up-regulated [154,157]. Depletion of ALYREF results in the reduced metastatic capacity ofhuman oral squamous cell carcinoma cell lines, demonstrating the increased TREX dependence in cancer cells.Interestingly, ALYREF depletion in this cell line results in increased levels of ‘metastasis modulating molecules’RRP1B and CD82 that suppress the metastatic capacity of tumor cells [158–160]. ALYREF directly interacts withRRP1B, so the increased expression of ALYREF in cancer cells may titrate away RRP1B allowing metastasis todevelop uninterrupted [157]. DDX39B depletion affects the expression levels of the tumor suppressor BRCA1protein, which is important for the DNA damage response [28]. SARNP is up-regulated in cancer tissues[37,161], but a further link to AML has been made attributable to a translocation event creating an SARNP–mixed lineage leukemia (MLL) fusion protein [162]. As MLL does not have any intrinsic DNA-binding capability,

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2925

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

the SARNP–MLL fusion may confer this action through SARNP’s SAP DNA-binding motif (Figure 2), potentiallyresulting in altered gene expression, a hallmark of cancer development [163].TREX seems to be directly involved in regulating the cell cycle, consistent with a role in proliferation and dif-

ferentiation, and this activity probably correlates with the aberrant expression of TREX subunits in cancer cells.During mitosis, the cell moves through characteristic stages and TREX may function at the intermediate check-points. THOC1 and THOC2 influence the rate of proliferation, with THOC1 playing a more specific role inthe G2-M checkpoint [50,54]. ALYREF is significant in the S and G2 phases where its expression seemsco-ordinated with that of Cyclin A [67]. ALYREF also represses the activity of the E2F2 transcription factorwhen bound to the CHK1 promoter, preventing the expression of the CHK1 checkpoint kinase that inducescell cycle arrest and checkpoint activation [164]. Furthermore, DDX39B and THOC2 may play a role inchromosome alignment [165] and spindle assembly [166], though whether these are direct effects or indirectthrough failure to export crucial mRNAs remains unclear.The numerous associations of TREX in cancer development could lead to new specific therapeutics. The

accumulating evidence that cancerous tissues are more dependent on TREX to aid their survival and progres-sion suggests that drugs to deplete TREX levels and/or activity could be a potential target for cancer therapy.The marked reduction in metastatic capacity and cancer cell proliferation following ALYREF [157], THOC1[21,153] and LUZP4 [19] depletion further suggests that there may be therapeutic benefit in targeting TREX.

Cellular differentiationIn addition to elevated TREX dependence in cancerous tissue, ES cells show a greater susceptibility to TREXloss. ES cells are pluripotent and derived from the inner cell mass (ICM) of the pre-implantation embryo atthe blastocyst stage. Depletion of THO components at this stage results in embryonic lethality as the blasto-cyst cannot be effectively formed due to increased apoptosis of the ICM [56,57,167]. However, depletion ofthese components in differentiated adult cells does not result in immediate cell death [168] and THOC1expression is decreased in nonproliferating cells [52], suggesting a greater dependency on TREX in undiffer-entiated cells early on in embryogenesis. Furthermore, the balance between differentiation and self-renewalwithin stem cells may be regulated by TREX through its function in mRNA export. Reduced levels ofTHOC2 and THOC5 during differentiation result in a decreased expression of self-renewal factors NANOGand SOX2, but an increased expression of differentiation markers GATA3 and BRACHURY [57,169]. THOC2and THOC5 associate with differentiation marker mRNAs more readily than self-renewal mRNAs and thenuclear accumulation of NANOG and SOX2 mRNA suggests that this regulation occurs through mRNAexport [57].The differentiation and development of specific tissues show a greater demand for TREX. The levels of

THOC5 and SARNP are greater in the human fetal liver, where hematopoietic cells differentiate [23], andSARNP is up-regulated in the adult bone marrow [37]. THOC5 is known to regulate differentiation of hemato-poietic cells to various lineages [55] and may do so by regulating the expression of specific transcription factorsinvolved in monocyte development [170]. The regulation of immediate early genes involved in macrophage dif-ferentiation is dependent on THOC5, perhaps linked to 30-end processing of the transcripts [112]. Bonemarrow and spleen cells are specifically affected following the loss of THOC5, resulting in death after a fewdays in mice [56,171], consistent with the loss of differentiating cells when THOC5 is depleted [172].Conversely, THOC5 is dispensable in the terminally differentiated liver [172]. The small intestine is remarkablysensitive to the loss of TREX, presumably due to the presence of stem-cell niches in the intestinal crypt.THOC1 expression is higher in intestinal crypts harboring stem-cell niches [168], and THOC1 depletionresults in degeneration of the small-intestinal epithelium and increased apoptosis of cells in the niche[168,172]. Interestingly, the stem-cell niches in the large intestine are not as affected by THOC1 depletion[168], demonstrating the tissue-specific effects of TREX loss as well as the increased requirement for thecomplex in differentiating tissues. Male gametogenesis requires rapid proliferation and stem-cell differentiationin the testes to produce sperm. Concurrent with increased TREX dependency in differentiating cells, fertility inmale mice was greatly reduced when two hypomorphic THOC1 alleles were present [173]. Female mice alsohad reduced fertility when homozygous for the hypomorphic allele, most probably due to defects in the ICMduring embryogenesis as mentioned above.From studies in cancer, embryogenesis and tissue differentiation, the dependence on TREX for cellular sur-

vival seems to be increased in more rapidly proliferating and differentiating cells. Proliferating cells will have ahigher rate of replication, transcription and therefore processing, export and translation to maintain cell

2926 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

division and induce differentiation. This increased burden on the gene expression machinery probably dictatesthe dependency on TREX that is central to this pathway.

Neurodevelopment and neurodegenerative diseasesThe role of TREX in neuronal disorders can be divided into two groups: direct mutations in TREX subunits,which trigger neuronal disease, and mutations in other genes, which lead to a potential dependence on TREXduring the disease state. In the first class, missense mutations in the THOC2 gene cause syndromic intellectualdisability [22]. Additionally, a de novo translocation event in the vicinity of THOC2 on the X-chromosome hasbeen described in a child with cerebellar hypoplasia, ataxia and retardation [54]. The translocation of the PTK2kinase gene generates a fusion transcript with THOC2 and leads to a decrease in expression of both encodedproteins. As inactivation of PTK2 alone does not cause this phenotype, the effects on THOC2 are likely to behugely influential in causing this disease, corroborated by C. elegans knockout studies showing impaired move-ment [54]. THOC6 mutations have also been associated with intellectual disability, consistent with its highexpression levels in the developing zebrafish brain [174]. The THOC6 mutation results in a mislocalization ofthe protein to the cytoplasm, suggesting that its nuclear role as a part of TREX is essential for proper neuraland organ development. Furthermore, a homozygous mutation in a conserved region of THOC6 is the likelycause of a disease with clinical features such as intellectual disability, brain malformation and renal and heartdefects [175].A second class of neuronal disease that indirectly depends on TREX is amyotrophic lateral sclerosis (ALS),

associated with death of motor neurons. The most common familial form of ALS involves a GGGGCC repeatexpansion within an intron of the C9ORF72 gene. Following transcription, the repeat expanded pre-mRNA isexported to the cytoplasm where it forms RNA foci. Thus, the transcript overrides the normal nuclear retentionmechanisms that keep pre-mRNA in the nucleus. The repeat expansion can also be translated in multiplereading frames in an ATG start codon-independent manner, leading to the production of toxic dipeptides(reviewed in ref. [176,177]). A survey of proteins that bind the repeat expansion identified multiple proteinsincluding ALYREF, leading to the suggestion that excess ALYREF recruitment to the repeat expandedpre-mRNA, may stimulate its export [178]. A Drosophila model for neurodegeneration triggered by theC9ORF72 was recently used to carry out a genetic screen for suppressors and enhancers of neurodegeneration[179]. This screen identified a mutation disrupting ALYREF activity as a potent suppressor of neurodegenera-tion in this system and intriguingly identified CHTOP and NXF1 as enhancers. Furthermore, it was shown thatthe toxic peptides that are produced by the repeat expansion block mRNA export in human cells [179].Therefore, in C9ORF72 disease, one unexpected reason for cell death may be the inhibition of expression ofmultiple proteins through a general mRNA export block. This export block is triggered by the inappropriateexport of the pre-mRNA containing the repeat and this may involve the excessive recruitment of ALYREF tothe repeat expansion.

Viral replicationViruses replicate by utilizing host cell machineries, which are essential for them to infect more cells and organ-isms. One such machinery is the NXF1–NXT1 mRNA export pathway that is employed to export viral mRNA,under the guise of a host mRNA. This ‘hijacking’ is conserved among viral species and subfamilies as it is usedin Influenza [32,180,181], various Herpes strains and the Hepatitis B virus. The mRNA export pathway is typic-ally hijacked by the expression of viral-specific export adaptors that serve to recruit TREX and NXF1 to viralmRNA. The Kaposi’s Sarcoma-associated Herpes Virus and Herpes virus saimiri ORF57 are the most wellstudied of these adaptors, recruiting ALYREF to the viral mRNA to which ORF57 is bound [182–185]. The restof TREX is also recruited, so the established RNA handover to NXF1–NXT1 can occur [184,186]. The Herpessimplex virus ICP27 [185,187], Epstein-Barr virus EB2 [188] and Human cytomegalovirus UL69 [189] proteinsare functionally homologous to ORF57 as they similarly recruit ALYREF, the rest of TREX and NXF1 for viralRNA export. In some cases, DDX39B seems to have more of an influence on viral RNA export than ALYREF[189,190], supporting the role of the whole TREX complex in viral mRNA export and efficient viral replication.In addition, ALYREF may act to stabilize viral mRNAs at the 30-end [191]. As well as viral export adaptors, ele-ments in viral RNA act to recruit the host cell mRNA export machinery. The posttranscriptional regulatoryelement is necessary for efficient expression of Hepatitis B proteins and triggers recruitment of TREX [44].Constitutive transport elements have been identified in a number of simple retroviruses, including MasonPfizer monkey and Simian retrovirus, which directly bind NXF1 with high affinity, promoting export of viral

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2927

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

transcripts [119]. Therefore, viruses have evolved a number of different strategies to hijack the cellular mRNAexport machinery to ensure efficient expression of their own proteins.

Plant developmentUnsurprisingly, TREX seems to play a role in other RNA pathways. The existence and general function ofTREX seems conserved in plants [8,75,192,193], and it also appears to play a role in the transport ofsmall-interfering RNA precursors for long-distance gene-silencing effects [8,194]. siRNA interaction with AGOmay require TREX to localize the siRNA, or to target the AGO protein necessary for gene silencing [8,194].THO mutants disrupt these pathways differentially depending on the plant’s developmental stage, consistentwith human systems showing a greater dependence on TREX during embryogenesis and in differentiatingtissues. TREX is implicated in other plant processes such as disease resistance and ethylene signaling, which isessential for development. Ethylene-induced senescence is increased in Hpr1 mutants, due to defective tran-scription of ethylene signaling and ribosomal protein genes [193,195]. The involvement of TREX in pathwaysother than mRNA export suggests that it may be a more general RNA trafficking module in the cell beyondplants, capable of influencing the localization of other RNA classes yet to be discovered.

DiscussionThe conserved TREX complex is highly integrated into cellular function and homeostasis (Figure 5), function-ing throughout mRNA biogenesis and in many other cellular mechanisms. Its presence is necessary for effectivetranscription, processing and export as well as preventing DNA damage and maintaining differentiation duringembryogenesis and in specific adult tissues. The dynamic nature of the proteins that associate with TREX islikely to provide the flexibility for the complex to carry out multiple functions and link mRNA generating andprocessing steps together. The less well-characterized members of TREX such as ERH, ZC3H11A andPOLDIP3 are likely to reveal further functions of TREX and refine the functions already known. A key andimportant step in understanding mammalian TREX function in the future will be to elucidate its RNA sub-strates, transcriptome-wide, as has recently been done for yeast TREX [92,99]. The additional functions ofTREX in the cell cycle, proliferating cells and the prevention of DNA damage must be investigated further,especially as understanding TREX could facilitate the development of novel drugs to treat cancer, ALS and viralinfections.

Figure 5. An overview of the biological processes involving TREX.

ESC: embryonic stem cell.

2928 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

AbbreviationsALS, amyotrophic lateral sclerosis; AML, acute myeloid leukemia; CBC, cap-binding complex; CTD, carboxyterminal domain; CTEs, constitutive transport elements; DDX39B, DEAD-box protein 39B; EJC, exon junctioncomplex; ES, embryonic stem; EPO, erythropoietin; ICM, inner cell mass; MLL, mixed lineage leukemia; mRNP,messenger ribonucleoprotein; NEXT, nuclear exosome targeting complex; NMD, nonsense mediated decay;NTF2L, NTF2-like; NXF1, nuclear export factor 1; Pol II, polymerase II; poly(A), polyadenosine A; pRB,retinoblastoma protein; PRMT1, protein arginine methyltransferase 1; RBD, RNA-binding domain; RNAi, RNAinterference; RRM, RNA recognition motif; TAP, tip-associated protein; TAR, transcription-associatedrecombination; Tip, tyrosine kinase interacting protein; TREX, TRanscription and EXport; UAP56,U2AF65-associated protein 56; UBM, UAP56-binding motif.

AcknowledgementsWe thank Steven West (Sheffield) for comments on the manuscript. S.A.W. acknowledges support from theBiotechnology and Biological Sciences Research Council, U.K.

Competing InterestsThe Authors declare that there are no competing interests associated with the manuscript.

References1 Topisirovic, I., Svitkin, Y.V., Sonenberg, N. and Shatkin, A.J. (2011) Cap and cap-binding proteins in the control of gene expression. Wiley Interdiscip.

Rev. 2, 277–298 doi:10.1002/wrna.522 Girard, C., Will, C.L., Peng, J., Makarov, E.M., Kastner, B., Lemm, I. et al. (2012) Post-transcriptional spliceosomes are retained in nuclear speckles

until splicing completion. Nat. Commun. 3, 994 doi:10.1038/ncomms19983 Proudfoot, N.J. (2011) Ending the message: poly(A) signals then and now. Genes Dev. 25, 1770–1782 doi:10.1101/gad.172684114 Kilchert, C., Wittmann, S. and Vasiljeva, L. (2016) The regulation and functions of the nuclear RNA exosome complex. Nat. Rev. Mol. Cell Biol. doi:10.

1038/nrm.2015.155 Trcek, T., Sato, H., Singer, R.H. and Maquat, L.E. (2013) Temporal and spatial characterization of nonsense-mediated mRNA decay. Genes Dev. 27,

541–551 doi:10.1101/gad.209635.1126 Strässer, K., Masuda, S., Mason, P., Pfannstiel, J., Oppizzi, M., Rodriguez-Navarro, S. et al. (2002) TREX is a conserved complex coupling transcription

with messenger RNA export. Nature 417, 304–308 doi:10.1038/nature7467 Rehwinkel, J., Herold, A., Gari, K., Köcher, T., Rode, M., Ciccarelli, F.L. et al. (2004) Genome-wide analysis of mRNAs regulated by the THO complex in

Drosophila melanogaster. Nat. Struct. Mol. Biol. 11, 558–566 doi:10.1038/nsmb7598 Yelina, N.E., Smith, L.M., Jones, A.M., Patel, K., Kelly, K.A. and Baulcombe, D.C. (2010) Putative Arabidopsis THO/TREX mRNA export complex is

involved in transgene and endogenous siRNA biosynthesis. Proc. Natl Acad. Sci. USA 107, 13948–13953 doi:10.1073/pnas.09113411079 El Bounkari, O., Guria, A., Klebba-Faerber, S., Claußen, M., Pieler, T., Griffiths, J.R. et al. (2009) Nuclear localization of the pre-mRNA associating

protein THOC7 depends upon its direct interaction with Fms tyrosine kinase interacting protein (FMIP). FEBS Lett. 583, 13–18 doi:10.1016/j.febslet.2008.11.024

10 Masuda, S., Das, R., Cheng, H., Hurt, E., Dorman, N. and Reed, R. (2005) Recruitment of the human TREX complex to mRNA during splicing. GenesDev. 19, 1512–1517 doi:10.1101/gad.1302205

11 Cheng, H., Dufu, K., Lee, C.S., Hsu, J.L., Dias, A. and Reed, R. (2006) Human mRNA export machinery recruited to the 50 end of mRNA. Cell 127,1389–1400 doi:10.1016/j.cell.2006.10.044

12 Gromadzka, A.M., Steckelberg, A.L., Singh, K.K., Hofmann, K. and Gehring, N.H. (2016) A short conserved motif in ALYREF directs cap- andEJC-dependent assembly of export complexes on spliced mRNAs. Nucleic Acids Res. doi:10.1093/nar/gkw009

13 Johnson, S.A., Cubberley, G. and Bentley, D.L. (2009) Cotranscriptional recruitment of the mRNA export factor Yra1 by direct interaction with the 30 endprocessing factor Pcf11. Mol. Cell 33, 215–226 doi:10.1016/j.molcel.2008.12.007

14 Dufu, K., Livingstone, M.J., Seebacher, J., Gygi, S.P., Wilson, S.A. and Reed, R. (2010) ATP is required for interactions between UAP56 and twoconserved mRNA export proteins, Aly and CIP29, to assemble the TREX complex. Genes Dev. 24, 2043–2053 doi:10.1101/gad.1898610

15 Chi, B., Wang, Q., Wu, G., Tan, M., Wang, L., Shi, M. et al. (2013) Aly and THO are required for assembly of the human TREX complex and associationof TREX components with the spliced mRNA. Nucleic Acids Res. 41, 1294–1306 doi:10.1093/nar/gks1188

16 Chang, C.-T., Hautbergue, G.M., Walsh, M.J., Viphakone, N., van Dijk, T.B., Philipsen, S. et al. (2013) Chtop is a component of the dynamic TREXmRNA export complex. EMBO J. 32, 473–486 doi:10.1038/emboj.2012.342

17 Hautbergue, G.M., Hung, M.L., Walsh, M.J., Snijders, A.P.L., Chang, C-T., Jones, R. et al. (2009) UIF, a new mRNA export adaptor that works togetherwith REF/ALY, requires FACT for recruitment to mRNA. Curr. Biol. 19, 1918–1924 doi:10.1016/j.cub.2009.09.041

18 Folco, E.G., Lee, C.S., Dufu, K., Yamazaki, T. and Reed, R. (2012) The proteins PDIP3 and ZC11A associate with the human TREX complex in anATP-dependent manner and function in mRNA export. PLoS ONE 7, e43804 doi:10.1371/journal.pone.0043804

19 Viphakone, N., Cumberbatch, M.G., Livingstone, M.J., Heath, P.R., Dickman, M.J., Catto, J.W. et al. (2015) Luzp4 defines a new mRNA export pathwayin cancer cells. Nucleic Acids Res. 43, 2353–2366 doi:10.1093/nar/gkv070

20 Durfee, T., Mancini, M.A., Jones, D., Elledge, S.J. and Lee, W.H. (1994) The amino-terminal region of the retinoblastoma gene product binds a novelnuclear matrix protein that co-localizes to centers for RNA processing. J. Cell Biol. 127, 609–622 doi:10.1083/jcb.127.3.609

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2929

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

21 Li, Y., Wang, X., Zhang, X. and Goodrich, D.W. (2005) Human hHpr1/p84/Thoc1 regulates transcriptional elongation and physically links RNApolymerase II and RNA processing factors. Mol. Cell. Biol. 25, 4023–4033 doi:10.1128/MCB.25.10.4023-4033.2005

22 Kumar, R., Corbett, M.A., van Bon, B.W.M., Woenig, J.A., Weir, L., Douglas, E. et al. (2015) THOC2 mutations implicate mRNA-export pathway inX-Linked intellectual disability. Am. J. Hum. Genet. 97, 302–310 doi:10.1016/j.ajhg.2015.05.021

23 Tamura, T., Mancini, A., Joos, H., Koch, A., Hakim, C., Dumanski, J. et al. (1999) FMIP, a novel Fms-interacting protein, affects granulocyte/macrophage differentiation. Oncogene 18, 6488–6495 doi:10.1038/sj.onc.1203062

24 Katahira, J., Inoue, H., Hurt, E. and Yoneda, Y. (2009) Adaptor Aly and co-adaptor Thoc5 function in the Tap-p15-mediated nuclear export of HSP70mRNA. EMBO J. 28, 556–567 doi:10.1038/emboj.2009.5

25 Fleckner, J., Zhang, M., Valcarcel, J. and Green, M.R. (1997) U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpointinteraction. Genes Dev. 11, 1864–1872 doi:10.1101/gad.11.14.1864

26 Shen, J., Zhang, L. and Zhao, R. (2007) Biochemical characterization of the ATPase and helicase activity of UAP56, an essential pre-mRNA splicing andmRNA export factor. J. Biol. Chem. 282, 22544–22550 doi:10.1074/jbc.M702304200

27 Pryor, A., Tung, L., Yang, Z., Kapadia, F., Chang, T.H. and Johnson, L.F. (2004) Growth-regulated expression and G0-specific turnover of the mRNA thatencodes URH49, a mammalian DExH/D box protein that is highly related to the mRNA export protein UAP56. Nucleic Acids Res. 32, 1857–1865doi:10.1093/nar/gkh347

28 Yamazaki, T., Fujiwara, N., Yukinaga, H., Ebisuya, M., Shiki, T., Kurihara, T. et al. (2010) The closely related RNA helicases, UAP56 and URH49, preferentiallyform distinct mRNA export machineries and coordinately regulate mitotic progression. Mol. Biol. Cell. 21, 2953–2965 doi:10.1091/mbc.E09-10-0913

29 Bruhn, L., Munnerlyn, A. and Grosschedl, R. (1997) ALY, a context-dependent coactivator of LEF-1 and AML-1, is required for TCRalpha enhancerfunction. Genes Dev. 11, 640–653 doi:10.1101/gad.11.5.640

30 Hautbergue, G.M., Hung, M.-L., Golovanov, A.P., Lian, L.-Y. and Wilson, S.A. (2008) Mutually exclusive interactions drive handover of mRNA from exportadaptors to TAP. Proc. Natl Acad. Sci. USA 105, 5154–5159 doi:10.1073/pnas.0709167105

31 Piruat, J.I. and Aguilera, A. (1998) A novel yeast gene, THO2, is involved in RNA pol II transcription and provides new evidence for transcriptionalelongation-associated recombination. EMBO J. 17, 4859–4872 doi:10.1093/emboj/17.16.4859

32 Hao, L., Sakurai, A., Watanabe, T., Sorensen, E., Nidom, C.A., Newton, M.A. et al. (2008) Drosophila RNAi screen identifies host genes important forinfluenza virus replication. Nature 454, 890–893 doi:10.1038/nature07151

33 Türeci, O., Sahin, U., Koslowski, M., Buss, B., Bell, C., Ballweber, P. et al. (2002) A novel tumour associated leucine zipper protein targeting to sites ofgene transcription and splicing. Oncogene 21, 3879–3888 doi:10.1038/sj.onc.1205481

34 Chavez, S., Beilharz, T., Rondon, A.G., Erdjument-Bromage, H., Tempst, P., Svejstrup, J.Q. et al. (2000) A protein complex containing Tho2, Hpr1, Mft1and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae. EMBO J. 19, 5824–5834doi:10.1093/emboj/19.21.5824

35 van Dijk, T.B., Gillemans, N., Stein, C., Fanis, P., Demmers, J., van de Corput, M. et al. (2010) Friend of Prmt1, a novel chromatin target of proteinarginine methyltransferases. Mol. Cell Biol. 30, 260–272 doi:10.1128/MCB.00645-09

36 Fanis, P., Gillemans, N., Aghajanirefah, A., Pourfarzad, F., Demmers, J., Esteghamat, F. et al. (2012) Five friends of methylated chromatin targetof protein-arginine-methyltransferase[prmt]-1 (chtop), a complex linking arginine methylation to desumoylation. Mol. Cell Proteomicsdoi:10.1074/mcp.M112.017194

37 Fukuda, S., Wu, D.W., Stark, K. and Pelus, L.M. (2002) Cloning and characterization of a proliferation-associated cytokine-inducible protein, CIP29.Biochem. Biophys. Res. Commun. 292, 593–600 doi:10.1006/bbrc.2002.6680

38 Richardson, C.J., Bröenstrup, M., Fingar, D.C., Jülich, K., Ballif, B.A., Gygi, S. et al. (2004) SKAR is a specific target of S6 kinase 1 in cell growthcontrol. Curr. Biol. 14, 1540–1549 doi:10.1016/j.cub.2004.08.061

39 Weng, M.T. and Luo, J. (2013) The enigmatic ERH protein: its role in cell cycle, RNA splicing and cancer. Protein Cell 4, 807–812 doi:10.1007/s13238-013-3056-3

40 Katahira, J., Sträßer, K., Podtelejnikov, A., Mann, M., Jung, J.U. and Hurt, E. (1999) The Mex67p-mediated nuclear mRNA export pathway is conservedfrom yeast to human. EMBO J. 18, 2593–2609 doi:10.1093/emboj/18.9.2593

41 Teplova, M., Wohlbold, L., Khin, N.W., Izaurralde, E. and Patel, D.J. (2011) Structure-function studies of nucleocytoplasmic transport of retroviralgenomic RNA by mRNA export factor TAP. Nat. Struct. Mol. Biol. 18, 990–998 doi:10.1038/nsmb.2094

42 Viphakone, N., Hautbergue, G.M., Walsh, M., Chang, C.-T., Holland, A., Folco, E.G. et al. (2012) TREX exposes the RNA-binding domain of Nxf1 toenable mRNA export. Nat. Commun. 3, 1006 doi:10.1038/ncomms2005

43 Fribourg, S., Braun, I.C., Izaurralde, E. and Conti, E. (2001) Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain ofthe TAP/p15 mRNA nuclear export factor. Mol. Cell. 8, 645–656 doi:10.1016/S1097-2765(01)00348-3

44 Chi, B., Wang, K., Du, Y., Gui, B., Chang, X., Wang, L. et al. (2014) A sub-element in PRE enhances nuclear export of intronless mRNAs by recruitingthe TREX complex via ZC3H18. Nucleic Acids Res. 42, 7305–7318 doi:10.1093/nar/gku350

45 Andersen, P.R., Domanski, M., Kristiansen, M.S., Storvall, H., Ntini, E., Verheggen, C. et al. (2013) The human cap-binding complex is functionallyconnected to the nuclear RNA exosome. Nat. Struct. Mol. Biol. 20, 1367–1376 doi:10.1038/nsmb.2703

46 Gebhardt, A., Habjan, M., Benda, C., Meiler, A., Haas, D.A., Hein, M.Y. et al. (2015) mRNA export through an additional cap-binding complex consistingof NCBP1 and NCBP3. Nat. Commun. 6, 8192 doi:10.1038/ncomms9192

47 Chavez, S. and Aguilera, A. (1997) The yeast HPR1 gene has a functional role in transcriptional elongation that uncovers a novel source of genomeinstability. Genes Dev. 11, 3459–3470 doi:10.1101/gad.11.24.3459

48 Schneiter, R., Guerra, C.E., Lampl, M., Gogg, G., Kohlwein, S.D. and Klein, H.L. (1999) The Saccharomyces cerevisiae hyperrecombination mutant hpr1Δ is synthetically lethal with two conditional alleles of the acetyl coenzyme A carboxylase gene and causes a defect in nuclear export of polyadenylatedRNA. Mol. Cell Biol. 19, 3415–3422 doi:10.1128/MCB.19.5.3415

49 Doostzadeh-Cizeron, J., Evans, R., Yin, S. and Goodrich, D.W. (1999) Apoptosis induced by the nuclear death domain protein p84N5 is inhibited byassociation with Rb protein. Mol. Biol. Cell. 10, 3251–3261 doi:10.1091/mbc.10.10.3251

50 Doostzadeh-Cizeron, J., Terry, N.H. and Goodrich, D.W. (2001) The nuclear death domain protein p84N5 activates a G2/M cell cycle checkpoint prior tothe onset of apoptosis. J. Biol. Chem. 276, 1127–1132 doi:10.1074/jbc.M006944200

2930 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

51 Evans, R.L., Poe, B.S. and Goodrich, D.W. (2002) Nuclear localization is required for induction of apoptotic cell death by the Rb-associated p84N5 deathdomain protein. Oncogene 21, 4691–4695 doi:10.1038/sj.onc.1205583

52 Gasparri, F., Sola, F., Locatelli, G. and Muzio, M. (2004) The death domain protein p84N5, but not the short isoform p84N5s, is cell cycle-regulatedand shuttles between the nucleus and the cytoplasm. FEBS Lett. 574, 13–19 doi:10.1016/j.febslet.2004.07.074

53 Peña, A., Gewartowski, K., Mroczek, S., Cuéllar, J., Szykowska, A., Prokop, A. et al. (2012) Architecture and nucleic acids recognition mechanism ofthe THO complex, an mRNP assembly factor. EMBO J. 31, 1605–1616 doi:10.1038/emboj.2012.10

54 Di Gregorio, E., Bianchi, F.T., Schiavi, A., Chiotto, A.M.A, Rolando, M., Verdun di Cantogno, L. et al. (2013) A de novo X;8 translocation creates aPTK2-THOC2 gene fusion with THOC2 expression knockdown in a patient with psychomotor retardation and congenital cerebellar hypoplasia.J. Med. Genet. 50, 543–551 doi:10.1136/jmedgenet-2013-101542

55 Mancini, A., Koch, A., Whetton, A.D. and Tamura, T. (2004) The M-CSF receptor substrate and interacting protein FMIP is governed in its subcellularlocalization by protein kinase C-mediated phosphorylation, and thereby potentiates M-CSF-mediated differentiation. Oncogene 23, 6581–6589doi:10.1038/sj.onc.1207841

56 Mancini, A., Niemann-Seyde, S.C., Pankow, R., El Bounkari, O., Klebba-Färber, S., Koch, A. et al. (2010) THOC5/FMIP, an mRNA export TREX complexprotein, is essential for hematopoietic primitive cell survival in vivo. BMC Biol. 8, 1 doi:10.1186/1741-7007-8-1

57 Wang, L., Miao, Y.-L., Zheng, X., Lackford, B., Zhou, B., Han, L. et al. (2013) The THO complex regulates pluripotency gene mRNA export and controlsembryonic stem cell self-renewal and somatic cell reprogramming. Cell Stem Cell 13, 676–690 doi:10.1016/j.stem.2013.10.008

58 Shen, H., Zheng, X., Shen, J., Zhang, L., Zhao, R. and Green, M.R. (2008) Distinct activities of the DExD/H-box splicing factor hUAP56 facilitatestepwise assembly of the spliceosome. Genes Dev. 22, 1796–1803 doi:10.1101/gad.1657308

59 Luo, M.-J., Zhou, Z., Magni, K., Christoforides, C., Rappsilber, J., Mann, M. et al. (2001) Pre-mRNA splicing and mRNA export linked by directinteractions between UAP56 and Aly. Nature 413, 644–647 doi:10.1038/35098106

60 Taniguchi, I. and Ohno, M. (2008) ATP-dependent recruitment of export factor Aly/REF onto intronless mRNAs by RNA helicase UAP56. Mol. Cell Biol.28, 601–608 doi:10.1128/MCB.01341-07

61 Kiesler, E., Miralles, F. and Visa, N. (2002) HEL/UAP56 binds cotranscriptionally to the Balbiani ring pre-mRNA in an intron-independent manner andaccompanies the BR mRNP to the nuclear pore. Curr. Biol. 12, 859–862 doi:10.1016/S0960-9822(02)00840-0

62 Virbasius, C.M., Wagner, S. and Green, M.R. (1999) A human nuclear-localized chaperone that regulates dimerization, DNA binding, and transcriptionalactivity of bZIP proteins. Mol. Cell. 4, 219–228 doi:10.1016/S1097-2765(00)80369-X

63 Golovanov, A.P., Hautbergue, G.M., Tintaru, A.M., Lian, L.-Y. and Wilson, S.A. (2006) The solution structure of REF2-I reveals interdomain interactionsand regions involved in binding mRNA export factors and RNA. RNA 12, 1933–1948 doi:10.1261/rna.212106

64 Portman, D.S., O’Connor, J.P. and Dreyfuss, G. (1997) YRA1, an essential Saccharomyces cerevisiae gene, encodes a novel nuclear protein with RNAannealing activity. RNA 3, 527–537 PMID: 9149233

65 Björk, P. and Wieslander, L. (2015) The Balbiani Ring Story: synthesis, assembly, processing, and transport of specific messenger RNA-proteincomplexes. Annu. Rev. Biochem. 84, 65–92 doi:10.1146/annurev-biochem-060614-034150

66 Hung, M.L., Hautbergue, G.M., Snijders, A.P., Dickman, M.J. and Wilson, S.A. (2010) Arginine methylation of REF/ALY promotes efficient handover ofmRNA to TAP/NXF1. Nucleic Acids Res. 38, 3351–3361 doi:10.1093/nar/gkq033

67 Okada, M., Jang, S.-W. and Ye, K. (2008) Akt phosphorylation and nuclear phosphoinositide association mediate mRNA export and cell proliferationactivities by ALY. Proc. Natl Acad. Sci. USA 105, 8649–8654 doi:10.1073/pnas.0802533105

68 Kim, V.N., Yong, J., Kataoka, N., Abel, L., Diem, M.D. and Dreyfuss, G. (2001) The Y14 protein communicates to the cytoplasm the position ofexon-exon junctions. EMBO J. 20, 2062–2068 doi:10.1093/emboj/20.8.2062

69 Rodrigues, J.P., Rode, M., Gatfield, D., Blencowe, B.J., Carmo-Fonseca, M. and Izaurralde, E. (2001) REF proteins mediate the export of spliced andunspliced mRNAs from the nucleus. Proc. Natl Acad. Sci. USA 98, 1030–1035 doi:10.1073/pnas.98.3.1030

70 Stubbs, S.H. and Conrad, N.K. (2015) Depletion of REF/Aly alters gene expression and reduces RNA polymerase II occupancy. Nucleic Acids Res. 43,504–519 doi:10.1093/nar/gku1278

71 Zullo, A.J., Michaud, M., Zhang, W. and Grusby, M.J. (2009) Identification of the small protein rich in arginine and glycine (SRAG): a newly identifiednucleolar protein that can regulate cell proliferation. J. Biol. Chem. 284, 12504–12511 doi:10.1074/jbc.M809436200

72 van Dijk, T.B., Gillemans, N., Pourfarzad, F., van Lom, K., von Lindern, M., Grosveld, F. et al. (2010) Fetal globin expression is regulated by friend ofPrmt1. Blood 116, 4349–4352 doi:10.1182/blood-2010-03-274399

73 Jimeno, S., Luna, R., Garcia-Rubio, M. and Aguilera, A. (2006) Tho1, a novel hnRNP, and Sub2 provide alternative pathways for mRNP biogenesis inyeast THO mutants. Mol. Cell Biol. 26, 4387–4398 doi:10.1128/MCB.00234-06

74 Sugiura, T., Sakurai, K. and Nagano, Y. (2007) Intracellular characterization of DDX39, a novel growth-associated RNA helicase. Exp. Cell Res. 313,782–790 doi:10.1016/j.yexcr.2006.11.014

75 Germain, H., Qu, N., Cheng, Y.T., Lee, E., Huang, Y., Dong, O.X. et al. (2010) MOS11: a new component in the mRNA export pathway. PLoS Genet. 6,e1001250 doi:10.1371/journal.pgen.1001250

76 Ma, X.M., Yoon, S.-O., Richardson, C.J., Jülich, K. and Blenis, J. (2008) SKAR links pre-mRNA splicing to mTOR/S6K1-mediated enhanced translationefficiency of spliced mRNAs. Cell 133, 303–313 doi:10.1016/j.cell.2008.02.031

77 Smyk, A., Szuminska, M., Uniewicz, K.A., Graves, L.M. and Kozlowski, P. (2006) Human enhancer of rudimentary is a molecular partner of PDIP46/SKAR,a protein interacting with DNA polymerase δ and S6K1 and regulating cell growth. FEBS J. 273, 4728–4741 doi:10.1111/j.1742-4658.2006.05477.x

78 Orphanides, G., Wu, W.H., Lane, W.S., Hampsey, M. and Reinberg, D. (1999) The chromatin-specific transcription elongation factor FACT compriseshuman SPT16 and SSRP1 proteins. Nature 400, 284–288 doi:10.1038/22350

79 Bortvin, A. and Winston, F. (1996) Evidence that Spt6p controls chromatin structure by a direct interaction with histones. Science 272, 1473–1476doi:10.1126/science.272.5267.1473

80 Yoh, S.M., Cho, H., Pickle, L., Evans, R.M. and Jones, K.A. (2007) The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicingand export. Genes Dev. 21, 160–174 doi:10.1101/gad.1503107

81 Meinel, D.M., Burkert-Kautzsch, C., Kieser, A., O’Duibhir, E., Siebert, M., Mayer, A. et al. (2013) Recruitment of TREX to the transcription machinery byits direct binding to the phospho-CTD of RNA polymerase II. PLoS Genet. 9, e1003914 doi:10.1371/journal.pgen.1003914

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2931

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

82 MacKellar, A.L. and Greenleaf, A.L. (2011) Cotranscriptional association of mRNA export factor Yra1 with C-terminal domain of RNA polymerase II.J. Biol. Chem. 286, 36385–36395 doi:10.1074/jbc.M111.268144

83 Gwizdek, C., Iglesias, N., Rodriguez, M.S., Ossareh-Nazari, B., Hobeika, M., Divita, G. et al. (2006) Ubiquitin-associated domain of Mex67 synchronizesrecruitment of the mRNA export machinery with transcription. Proc. Natl Acad. Sci. USA 103, 16376–16381 doi:10.1073/pnas.0607941103

84 Chanarat, S. and Sträßer, K. (2013) Splicing and beyond: the many faces of the Prp19 complex. Biochim. Biophys. Acta 1833, 2126–2134doi:10.1016/j.bbamcr.2013.05.023

85 Chanarat, S., Seizl, M. and Strasser, K. (2011) The Prp19 complex is a novel transcription elongation factor required for TREX occupancy at transcribedgenes. Genes Dev. 25, 1147–1158 doi:10.1101/gad.623411

86 David, C.J., Boyne, A.R., Millhouse, S.R. and Manley, J.L. (2011) The RNA polymerase II C-terminal domain promotes splicing activation throughrecruitment of a U2AF65-Prp19 complex. Genes Dev. 25, 972–983 doi:10.1101/gad.2038011

87 Dominguez-Sánchez, M.S., Barroso, S., Gómez-González, B., Luna, R. and Aguilera, A. (2011) Genome instability and transcription elongationimpairment in human cells depleted of THO/TREX. PLoS Genet. 7, e1002386| doi:10.1371/journal.pgen.1002386

88 Davidson, L., Muniz, L. and West, S. (2014) 30 end formation of pre-mRNA and phosphorylation of Ser2 on the RNA polymerase II CTD are reciprocallycoupled in human cells. Genes Dev. 28, 342–356 doi:10.1101/gad.231274.113

89 McCloskey, A., Taniguchi, I., Shinmyozu, K. and Ohno, M. (2012) hnRNP C tetramer measures RNA length to classify RNA polymerase II transcripts forexport. Science 335, 1643–1646 doi:10.1126/science.1218469

90 Luo, M.-J. and Reed, R. (1999) Splicing is required for rapid and efficient mRNA export in metazoans. Proc. Natl Acad. Sci. USA 96, 14937–14942doi:10.1073/pnas.96.26.14937

91 Takemura, R., Takeiwa, T., Taniguchi, I., McCloskey, A. and Ohno, M. (2011) Multiple factors in the early splicing complex are involved in the nuclearretention of pre-mRNAs in mammalian cells. Genes Cells doi:10.1111/j.1365-2443.2011.01548

92 Baejen, C., Torkler, P., Gressel, S., Essig, K., Söding, J. and Cramer, P. (2014) Transcriptome maps of mRNP biogenesis factors define pre-mRNArecognition. Mol. Cell. 55, 745–757 doi:10.1016/j.molcel.2014.08.005

93 Teng, I.-F. and Wilson, S.A. (2013) Mapping interactions between mRNA export factors in living cells. PLoS ONE 8, e67676 doi:10.1371/journal.pone.0067676

94 Dias, A.P., Dufu, K., Lei, H. and Reed, R. (2010) A role for TREX components in the release of spliced mRNA from nuclear speckle domains. Nat.Commun. 1, 97 doi:10.1038/ncomms1103

95 Le Hir, H., Izaurralde, E., Maquat, L.E. and Moore, M.J. (2000) The spliceosome deposits multiple proteins 20–24 nucleotides upstream of mRNAexon-exon junctions. EMBO J. 19, 6860–6869 doi:10.1093/emboj/19.24.6860

96 Le Hir, H., Gatfield, D., Izaurralde, E. and Moore, M.J. (2001) The exon-exon junction complex provides a binding platform for factors involved in mRNAexport and nonsense-mediated mRNA decay. EMBO J. 20, 4987–4997 doi:10.1093/emboj/20.17.4987

97 Singh, G., Kucukural, A., Cenik, C., Leszyk, J.D., Shaffer, S.A., Weng, Z. et al. (2012) The cellular EJC interactome reveals higher-order mRNP structureand an EJC-SR protein nexus. Cell 151, 750–764 doi:10.1016/j.cell.2012.10.007

98 Daguenet, E., Baguet, A., Degot, S., Schmidt, U., Alpy, F., Wendling, C. et al. (2012) Perispeckles are major assembly sites for the exon junction corecomplex. Mol. Biol. Cell. 23, 1765–1782 doi:10.1091/mbc.E12-01-0040

99 Tuck, A.C. and Tollervey, D. (2013) A transcriptome-wide atlas of RNP composition reveals diverse classes of mRNAs and lncRNAs. Cell 154,996–1009 doi:10.1016/j.cell.2013.07.047

100 Kaida, D., Motoyoshi, H., Tashiro, E., Nojima, T., Hagiwara, M., Ishigami, K. et al. (2007) Spliceostatin A targets SF3b and inhibits both splicing andnuclear retention of pre-mRNA. Nat. Chem. Biol. 3, 576–583 doi:10.1038/nchembio.2007.18

101 Martins, S.B., Rino, J., Carvalho, T., Carvalho, C., Yoshida, M., Klose, J.M. et al. (2011) Spliceosome assembly is coupled to RNA polymerase IIdynamics at the 30 end of human genes. Nat. Struct. Mol. Biol. 18, 1115–1123 doi:10.1038/nsmb.2124

102 Nojima, T., Hirose, T., Kimura, H. and Hagiwara, M. (2007) The interaction between cap-binding complex and RNA export factor is required for intronlessmRNA export. J. Biol. Chem. 282, 15645–15651 doi:10.1074/jbc.M700629200

103 Blanchette, M., Labourier, E., Green, R.E., Brenner, S.E. and Rio, D.C. (2004) Genome-wide analysis reveals an unexpected function for the Drosophilasplicing factor U2AF50 in the nuclear export of intronless mRNAs. Mol. Cell. 14, 775–786 doi:10.1016/j.molcel.2004.06.012

104 Lei, H., Dias, A.P. and Reed, R. (2011) Export and stability of naturally intronless mRNAs require specific coding region sequences and the TREX mRNAexport complex. Proc. Natl Acad. Sci. USA 108, 17985–17990 doi:10.1073/pnas.1113076108

105 Lei, H., Zhai, B., Yin, S., Gygi, S. and Reed, R. (2013) Evidence that a consensus element found in naturally intronless mRNAs promotes mRNA export.Nucleic Acids Res. 41, 2517–2525 doi:10.1093/nar/gks1314

106 Huang, Y. and Steitz, J.A. (2001) Splicing factors SRp20 and 9G8 promote the nucleocytoplasmic export of mRNA. Mol. Cell. 7, 899–905doi:10.1016/S1097-2765(01)00233-7

107 Hargous, Y., Hautbergue, G.M., Tintaru, A.M., Skrisovska, L., Golovanov, A.P., Stevenin, J. et al. (2006) Molecular basis of RNA recognition and TAPbinding by the SR proteins SRp20 and 9G8. EMBO J. 25, 5126–5137 doi:10.1038/sj.emboj.7601385

108 Tintaru, A.M., Hautbergue, G.M., Hounslow, A.M., Hung, M.-L., Lian, L.-Y., Craven, C.J. et al. (2007) Structural and functional analysis of RNA and TAPbinding to SF2/ASF. EMBO Rep. 8, 756–762 doi:10.1038/sj.embor.7401031

109 Johnson, S.A., Kim, H., Erickson, B. and Bentley, D.L. (2011) The export factor Yra1 modulates mRNA 30 end processing. Nat. Struct. Mol. Biol. 18,1164–1171 doi:10.1038/nsmb.2126

110 Rougemaille, M., Dieppois, G., Kisseleva-Romanova, E., Gudipati, R.K., Lemoine, S., Blugeon, C. et al. (2008) THO/sub2p functions to coordinate 30-endprocessing with gene-nuclear pore association. Cell 135, 308–321 doi:10.1016/j.cell.2008.08.005

111 Shi, Y., Di Giammartino, D.C., Taylor, D., Sarkeshik, A., Rice, W.J., Yates, III, J.R. et al. (2009) Molecular architecture of the human pre-mRNA 30

processing complex. Mol. Cell. 33, 365–376 doi:10.1016/j.molcel.2008.12.028112 Tran, D.D.H., Saran, S., Williamson, A.J.K., Pierce, A., Dittrich-Breiholz, O., Wiehlmann, L. et al. (2014) THOC5 controls 30 end-processing of immediate

early genes via interaction with polyadenylation specific factor 100 (CPSF100). Nucleic Acids Res. 42, 12249–12260 doi:10.1093/nar/gku911113 Katahira, J., Okuzaki, D., Inoue, H., Yoneda, Y., Maehara, K. and Ohkawa, Y. (2013) Human TREX component Thoc5 affects alternative polyadenylation

site choice by recruiting mammalian cleavage factor I. Nucleic Acids Res. 41, 7060–7072 doi:10.1093/nar/gkt414

2932 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

114 Pak, V., Eifler, T.T., Jäger, S., Krogan, N.J., Fujinaga, K. and Peterlin, B.M. (2015) CDK11 in TREX/THOC regulates HIV mRNA 30 end processing. CellHost Microbe 18, 560–570 doi:10.1016/j.chom.2015.10.012

115 Schwanhäusser, B., Busse, D., Li, N., Dittmar, G., Schuchhardt, J., Wolf, J. et al. (2011) Global quantification of mammalian gene expression control.Nature 473, 337–342 doi:10.1038/nature10098

116 Gómez-González, B., García-Rubio, M., Bermejo, R., Gaillard, H., Shirahige, K., Marín, A. et al. (2011) Genome-wide function of THO/TREX in activegenes prevents R-loop-dependent replication obstacles. EMBO J. 30, 3106–3119 doi:10.1038/emboj.2011.206

117 Yoon, D.W., Lee, H., Seol, W., DeMaria, M., Rosenzweig, M. and Jung, J.U. (1997) Tap: a novel cellular protein that interacts with tip of Herpesvirussaimiri and induces lymphocyte aggregation. Immunity 6, 571–582 doi:10.1016/S1074-7613(00)80345-3

118 Segref, A., Sharma, K., Doye, V., Hellwig, A., Huber, J., Luhrmann, R. et al. (1997) Mex67p, a novel factor for nuclear mRNA export, binds to both poly(A)+ RNA and nuclear pores. EMBO J. 16, 3256–3271 doi:10.1093/emboj/16.11.3256

119 Grüter, P., Tabernero, C., von Kobbe, C., Schmitt, C., Saavedra, C., Bachi, A. et al. (1998) TAP, the human homolog of Mex67p, mediatesCTE-dependent RNA export from the nucleus. Mol. Cell. 1, 649–659 doi:10.1016/S1097-2765(00)80065-9

120 Liker, E., Fernandez, E., Izaurralde, E. and Conti, E. (2000) The structure of the mRNA export factor TAP reveals a cis arrangement of a non-canonicalRNP domain and an LRR domain. EMBO J. 19, 5587–5598 doi:10.1093/emboj/19.21.5587

121 Aibara, S., Katahira, J., Valkov, E. and Stewart, M. (2015) The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platformthat facilitates export of retroviral CTE-RNA. Nucleic Acids Res. 43, 1883–1893 doi:10.1093/nar/gkv032

122 Wickramasinghe, V.O., McMurtrie, P.I.A, Mills, A.D., Takei, Y., Penrhyn-Lowe, S., Amagase, Y. et al. (2010) mRNA export from mammalian cell nuclei isdependent on GANP. Curr. Biol. 20, 25–31 doi:10.1016/j.cub.2009.10.078

123 Jani, D., Lutz, S., Hurt, E., Laskey, R.A., Stewart, M. and Wickramasinghe, V.O. (2012) Functional and structural characterization of the mammalianTREX-2 complex that links transcription with nuclear messenger RNA export. Nucleic Acids Res. 40, 4562–4573 doi:10.1093/nar/gks059

124 Umlauf, D., Bonnet, J., Waharte, F., Fournier, M., Stierle, M., Fischer, B. et al. (2013) The human TREX-2 complex is stably associated with the nuclearpore basket. J. Cell Sci. 126, 2656–2667 doi:10.1242/jcs.118000

125 Schneider, M., Hellerschmied, D., Schubert, T., Amlacher, S., Vinayachandran, V., Reja, R. et al. (2015) The nuclear pore-associated TREX-2 complexemploys mediator to regulate gene expression. Cell 162, 1016–1028 doi:10.1016/j.cell.2015.07.059

126 Rodrı´guez-Navarro, S., Fischer, T., Luo, M.-J., Antúnez, O., Brettschneider, S., Lechner, J. et al. (2004) Sus1, a functional component of the SAGAhistone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 116, 75–86 doi:10.1016/S0092-8674(03)01025-0

127 Wickramasinghe, V.O., Andrews, R., Ellis, P., Langford, C., Gurdon, J.B., Stewart, M. et al. (2014) Selective nuclear export of specific classes of mRNAfrom mammalian nuclei is promoted by GANP. Nucleic Acids Res. 42, 5059–5071 doi:10.1093/nar/gku095

128 Wickramasinghe, V.O. and Laskey, R.A. (2015) Control of mammalian gene expression by selective mRNA export. Nat. Rev. Mol. Cell Biol. 16, 431–442doi:10.1038/nrm4010

129 Zolotukhin, A.S., Tan, W., Bear, J., Smulevitch, S. and Felber, B.K. (2002) U2AF participates in the binding of TAP (NXF1) to mRNA. J. Biol. Chem.277, 3935–3942 doi:10.1074/jbc.M107598200

130 Katahira, J., Dimitrova, L., Imai, Y. and Hurt, E. (2015) NTF2-like domain of Tap plays a critical role in cargo mRNA recognition and export. NucleicAcids Res. 43, 1894–1904 doi:10.1093/nar/gkv039

131 Li, Y., Bor, Y.-C., Misawa, Y., Xue, Y., Rekosh, D. and Hammarskjöld, M.-L. (2006) An intron with a constitutive transport element is retained in a Tapmessenger RNA. Nature 443, 234–237 doi:10.1038/nature05107

132 Huang, Y., Gattoni, R., Stévenin, J. and Steitz, J.A. (2003) SR splicing factors serve as adapter proteins for TAP-dependent mRNA export. Mol. Cell. 11,837–843 doi:10.1016/S1097-2765(03)00089-3

133 Hurt, E., Luo, M.-J., Röther, S., Reed, R. and Sträßer, K. (2004) Cotranscriptional recruitment of the serine-arginine-rich (SR)-like proteins Gbp2 andHrb1 to nascent mRNA via the TREX complex. Proc. Natl Acad. Sci. USA 101, 1858–1862 doi:10.1073/pnas.0308663100

134 Reed, R. and Cheng, H. (2005) TREX, SR proteins and export of mRNA. Curr. Opin. Cell Biol. 17, 269–273 doi:10.1016/j.ceb.2005.04.011135 Martínez-Lumbreras, S., Taverniti, V., Zorrilla, S., Séraphin, B. and Pérez-Cañadillas, J.M. (2016) Gbp2 interacts with THO/TREX through a novel type of

RRM domain. Nucleic Acids Res. 44, 437–448 doi:10.1093/nar/gkv1303136 Lindtner, S., Zolotukhin, A.S., Uranishi, H., Bear, J., Kulkarni, V., Smulevitch, S. et al. (2006) RNA-binding motif protein 15 binds to the RNA transport

element RTE and provides a direct link to the NXF1 export pathway. J. Biol. Chem. 281, 36915–36928 doi:10.1074/jbc.M608745200137 Ruepp, M.-D., Aringhieri, C., Vivarelli, S., Cardinale, S., Paro, S., Schumperli, D. et al. (2009) Mammalian pre-mRNA 30 end processing factor CF Im 68

functions in mRNA export. Mol. Biol. Cell. 20, 5211–5223 doi:10.1091/mbc.E09-05-0389138 Qu, X., Lykke-Andersen, S., Nasser, T., Saguez, C., Bertrand, E., Jensen, T.H. et al. (2009) Assembly of an export-competent mRNP is needed for

efficient release of the 30-end processing complex after polyadenylation. Mol. Cell Biol. 29, 5327–5338 doi:10.1128/MCB.00468-09139 Hurt, J.A., Obar, R.A., Zhai, B., Farny, N.G., Gygi, S.P. and Silver, P.A. (2009) A conserved CCCH-type zinc finger protein regulates mRNA nuclear

adenylation and export. J. Cell Biol. 185, 265–277 doi:10.1083/jcb.200811072140 Folkmann, A.W., Noble, K.N., Cole, C.N. and Wente, S.R. (2011) Dbp5, Gle1-IP6, and Nup159: a working model for mRNP export. Nucleus 2, 540–548

doi:10.4161/nucl.2.6.17881141 Schmitt, C., von Kobbe, C., Bachi, A., Pante, N., Rodrigues, J.P., Boscheron, C. et al. (1999) Dbp5, a DEAD-box protein required for mRNA export, is

recruited to the cytoplasmic fibrils of nuclear pore complex via a conserved interaction with CAN/Nup159p. EMBO J. 18, 4332–4347 doi:10.1093/emboj/18.15.4332

142 Folkmann, A.W., Collier, S.E., Zhan, X., Aditi, X., Ohi, M.D. and Wente, S.R. (2013) Gle1 functions during mRNA export in an oligomeric complex that isaltered in human disease. Cell 155, 582–593 doi:10.1016/j.cell.2013.09.023

143 Zhang, F., Wang, J., Xu, J., Zhang, Z., Koppetsch, B.S., Schultz, N. et al. (2012) UAP56 couples piRNA clusters to the perinuclear transposon silencingmachinery. Cell 151, 871–884 doi:10.1016/j.cell.2012.09.040

144 Hur, J.K., Luo, Y., Moon, S., Ninova, M., Marinov, G.K., Chung, Y.D. et al. (2016) Splicing-independent loading of TREX on nascent RNA is required forefficient expression of dual-strand piRNA clusters in Drosophila. Genes Dev. 30, 840–855 doi:10.1101/gad.276030.115

145 Santos-Pereira, J.M. and Aguilera, A. (2015) R loops: new modulators of genome dynamics and function. Nat. Rev. Genet. 16, 583–597doi:10.1038/nrg3961

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2933

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

146 Wickramasinghe, V.O. and Venkitaraman, A.R. (2016) RNA processing and genome stability: cause and consequence. Mol. Cell. 61, 496–505doi:10.1016/j.molcel.2016.02.001

147 Huertas, P. and Aguilera, A. (2003) Cotranscriptionally formed DNA:RNA hybrids mediate transcription elongation impairment andtranscription-associated recombination. Mol. Cell. 12, 711–721 doi:10.1016/j.molcel.2003.08.010

148 Paulsen, R.D., Soni, D.V., Wollman, R., Hahn, A.T., Yee, M.-C., Guan, A. et al. (2009) A genome-wide siRNA screen reveals diverse cellular processesand pathways that mediate genome stability. Mol. Cell. 35, 228–239 doi:10.1016/j.molcel.2009.06.021

149 Nedea, E., Nalbant, D., Xia, D., Theoharis, N.T., Suter, B., Richardson, C.J. et al. (2008) The Glc7 phosphatase subunit of the cleavage andpolyadenylation factor is essential for transcription termination on snoRNA genes. Mol. Cell. 29, 577–587 doi:10.1016/j.molcel.2007.12.031

150 Wickramasinghe, V.O., Savill, J.M., Chavali, S., Jonsdottir, A.B., Rajendra, E., Grüner, T. et al. (2013) Human inositol polyphosphate multikinaseregulates transcript-selective nuclear mRNA export to preserve genome integrity. Mol. Cell. 51, 737–750 doi:10.1016/j.molcel.2013.08.031

151 Takai, H., Masuda, K., Sato, T., Sakaguchi, Y., Suzuki, T., Suzuki, T. et al. (2014) 5-Hydroxymethylcytosine plays a critical role in glioblastomagenesisby recruiting the CHTOP-methylosome complex. Cell Rep. 9, 48–60 doi:10.1016/j.celrep.2014.08.071

152 Li, Y., Lin, A.W., Zhang, X., Wang, Y., Wang, X. and Goodrich, D.W. (2007) Cancer cells and normal cells differ in their requirements for Thoc1.Cancer Res. 67, 6657–6664 doi:10.1158/0008-5472.CAN-06-3234

153 Guo, S., Hakimi, M.A., Baillat, D., Chen, X., Farber, M.J., Klein-Szanto, A.J. et al. (2005) Linking transcriptional elongation and messenger RNA exportto metastatic breast cancers. Cancer Res. 65, 3011–3016 PMID: 15833825

154 Domínguez-Sánchez, M.S., Sáez, C., Japón, M.A., Aguilera, A. and Luna, R. (2011) Differential expression of THOC1 and ALY mRNP biogenesis/exportfactors in human cancers. BMC Cancer 11, 77 doi:10.1186/1471-2407-11-77

155 Guo, S., Liu, M. and Godwin, A.K. (2012) Transcriptional regulation of hTREX84 in human cancer cells. PLoS ONE 7, e43610 doi:10.1371/journal.pone.0043610

156 Griaud, F., Pierce, A., Gonzalez Sanchez, M.B., Scott, M., Abraham, S.A., Holyoake, T.L. et al. (2013) A pathway from leukemogenic oncogenes andstem cell chemokines to RNA processing via THOC5. Leukemia 27, 932–940 doi:10.1038/leu.2012.283

157 Saito, Y., Kasamatsu, A., Yamamoto, A., Shimizu, T., Yokoe, H., Sakamoto, Y. et al. (2013) ALY as a potential contributor to metastasis in human oralsquamous cell carcinoma. J. Cancer Res. Clin. Oncol. 139, 585–594 doi:10.1007/s00432-012-1361-5

158 Crawford, N.P.S., Qian, X., Ziogas, A., Papageorge, A.G., Boersma, B.J., Walker, R.C. et al. (2007) Rrp1b, a new candidate susceptibility gene forbreast cancer progression and metastasis. PLoS Genet. 3, e214 doi:10.1371/journal.pgen.0030214

159 Crawford, N.P., Yang, H., Mattaini, K.R. and Hunter, K.W. (2009) The metastasis efficiency modifier ribosomal RNA processing 1 homolog B (RRP1B)is a chromatin-associated factor. J. Biol. Chem. 284, 28660–28673 doi:10.1074/jbc.M109.023457

160 Tsai, Y.C. and Weissman, A.M. (2011) Dissecting the diverse functions of the metastasis suppressor CD82/KAI1. FEBS Lett. 585, 3166–3173doi:10.1016/j.febslet.2011.08.031

161 Choong, M.L., Tan, L.K., Lo, S.L., Ren, E.-C., Ou, K., Ong, S.-E. et al. (2001) An integrated approach in the discovery and characterization of a novelnuclear protein over-expressed in liver and pancreatic tumors. FEBS Lett. 496, 109–116 doi:10.1016/S0014-5793(01)02409-7

162 Hashii, Y., Kim, J.Y., Sawada, A., Tokimasa, S., Hiroyuki, F., Ohta, H. et al. (2004) A novel partner gene CIP29 containing a SAP domain with MLLidentified in infantile myelomonocytic leukemia. Leukemia 18, 1546–1548 doi:10.1038/sj.leu.2403450

163 Li, B.E. and Ernst, P. (2014) Two decades of leukemia oncoprotein epistasis: the MLL1 paradigm for epigenetic deregulation in leukemia. Exp. Hematol.42, 995–1012 doi:10.1016/j.exphem.2014.09.006

164 Osinalde, N., Olea, M., Mitxelena, J., Aloria, K., Rodriguez, J.A., Fullaondo, A. et al. (2013) The nuclear protein ALY binds to and modulates the activityof transcription factor E2F2. Mol. Cell. Proteomics 12, 1087–1098 doi:10.1074/mcp.M112.024158

165 Goshima, G., Wollman, R., Goodwin, S.S., Zhang, N., Scholey, J.M., Vale, R.D. et al. (2007) Genes required for mitotic spindle assembly in DrosophilaS2 cells. Science 316, 417–421 doi:10.1126/science.1141314

166 Somma, M.P., Ceprani, F., Bucciarelli, E., Naim, V., De Arcangelis, V., Piergentili, R. et al. (2008) Identification of Drosophila mitotic genes by combiningco-expression analysis and RNA interference. PLoS Genet. 4, e1000126 doi:10.1371/journal.pgen.1000126

167 Wang, X., Chang, Y., Li, Y., Zhang, X. and Goodrich, D.W. (2006) Thoc1/Hpr1/p84 is essential for early embryonic development in the mouse. Mol. Cell.Biol. 26, 4362–4367 doi:10.1128/MCB.02163-05

168 Pitzonka, L., Wang, X., Ullas, S., Wolff, D.W., Wang, Y. and Goodrich, D.W. (2013) The THO ribonucleoprotein complex is required for stem cellhomeostasis in the adult mouse small intestine. Mol. Cell. Biol. 33, 3505–3514 doi:10.1128/MCB.00751-13

169 Hu, G., Kim, J., Xu, Q., Leng, Y., Orkin, S.H. and Elledge, S.J. (2009) A genome-wide RNAi screen identifies a new transcriptional module required forself-renewal. Genes Dev. 23, 837–848 doi:10.1101/gad.1769609

170 Carney, L., Pierce, A., Rijnen, M., Gonzalez Sanchez, M.B., Hamzah, H.G., Zhang, L. et al. (2009) THOC5 couples M-CSF receptor signaling totranscription factor expression. Cell Signal. 21, 309–316 doi:10.1016/j.cellsig.2008.10.018

171 Tran, D.D.H., Saran, S., Dittrich-Breiholz, O., Williamson, A.J.K., Klebba-Färber, S., Koch, A. et al. (2013) Transcriptional regulation of immediate-earlygene response by THOC5, a member of mRNA export complex, contributes to the M-CSF-induced macrophage differentiation. Cell Death Dis. 4, e879doi:10.1038/cddis.2013.409

172 Saran, S., Tran, D.D.H., Klebba-Färber, S., Moran-Losada, P., Wiehlmann, L., Koch, A. et al. (2013) THOC5, a member of the mRNA export complex,contributes to processing of a subset of wingless/integrated (Wnt) target mRNAs and integrity of the gut epithelial barrier. BMC Cell Biol. 14, 51doi:10.1186/1471-2121-14-51

173 Wang, X., Chinnam, M., Wang, J., Wang, Y., Zhang, X., Marcon, E. et al. (2009) Thoc1 deficiency compromises gene expression necessary for normaltestis development in the mouse. Mol. Cell. Biol. 29, 2794–2803 doi:10.1128/MCB.01633-08

174 Beaulieu, C.L., Huang, L., Innes, A, Akimenko, M.-A., Puffenberger, E.G., Schwartz, C. et al. (2013) Intellectual disability associated with a homozygousmissense mutation in THOC6. Orphanet J. Rare Dis. 8, 62 doi:10.1186/1750-1172-8-62

175 Boycott, K.M., Beaulieu, C., Puffenberger, E.G. McLeod, D.R., Parboosingh, J.S. and Innes, A.M. (2010) A novel autosomal recessive malformationsyndrome associated with developmental delay and distinctive facies maps to 16ptel in the Hutterite population. Am. J. Med. Genet. 152A, 1349–1356doi:10.1002/ajmg.a.33379

2934 © 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY)

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010

176 Walsh, M.J., Cooper-Knock, J., Dodd, J.E., Stopford, M.J., Mihaylov, S.R., Kirby, J. et al. (2015) Invited review: decoding the pathophysiologicalmechanisms that underlie RNA dysregulation in neurodegenerative disorders: a review of the current state of the art. Neuropathol. Appl. Neurobiol. 41,109–134 doi:10.1111/nan.12187

177 Rohrer, J.D., Isaacs, A.M., Mizielinska, S., Mead, S., Lashley, T., Wray, S. et al. (2015) C9orf72 expansions in frontotemporal dementia and amyotrophiclateral sclerosis. Lancet Neurol. 14, 291–301 doi:10.1016/S1474-4422(14)70233-9

178 Cooper-Knock, J., Walsh, M.J., Higginbottom, A., Robin Highley, J., Dickman, M.J., Edbauer, D. et al. (2014) Sequestration of multiple RNA recognitionmotif-containing proteins by C9orf72 repeat expansions. Brain 137, 2040–2051 doi:10.1093/brain/awu120

179 Freibaum, B.D., Lu, Y., Lopez-Gonzalez, R., Kim, N.C., Almeida, S., Lee, K.H. et al. (2015) GGGGCC repeat expansion in C9orf72 compromisesnucleocytoplasmic transport. Nature doi:10.1038/nature14974

180 Read, E.K. and Digard, P. (2010) Individual influenza A virus mRNAs show differential dependence on cellular NXF1/TAP for their nuclear export.J. Gen. Virol. 91, 1290–1301 doi:10.1099/vir.0.018564-0

181 Larsen, S., Bui, S., Perez, V., Mohammad, A., Medina-Ramirez, H. and Newcomb, L.L. (2014) Influenza polymerase encoding mRNAs utilize atypicalmRNA nuclear export. Virol. J. 11, 154 doi:10.1186/1743-422X-11-154

182 Malik, P., Blackbourn, D.J. and Clements, J.B. (2004) The evolutionarily conserved Kaposi’s sarcoma-associated Herpesvirus ORF57 protein interactswith REF protein and acts as an RNA export factor. J. Biol. Chem. 279, 33001–33011 doi:10.1074/jbc.M313008200

183 Williams, B.J.L, Boyne, J.R., Goodwin, D.J., Roaden, L., Hautbergue, G.M., Wilson, S.A. et al. (2005) The prototype gamma-2 herpesvirusnucleocytoplasmic shuttling protein, ORF 57, transports viral RNA via the cellular mRNA export pathway. Biochem. J. 387, 295–308doi:10.1042/BJ20041223

184 Boyne, J.R., Colgan, K.J. and Whitehouse, A. (2008) Recruitment of the complete hTREX complex is required for Kaposi’s sarcoma-associatedHerpesvirus intronless mRNA nuclear export and virus replication. PLoS Pathog. 4, e1000194 doi:10.1371/journal.ppat.1000194

185 Tunnicliffe, R.B., Hautbergue, G.M., Kalra, P., Jackson, B.R., Whitehouse, A., Wilson, S.A. et al. (2011) Structural basis for the recognition of cellularmRNA export factor REF by herpes viral proteins HSV-1 ICP27 and HVS ORF57. PLoS Pathog. 7, e1001244 doi:10.1371/journal.ppat.1001244

186 Jackson, B.R., Boyne, J.R., Noerenberg, M., Taylor, A., Hautbergue, G.M., Walsh, M.J. et al. (2011) An interaction between KSHV ORF57 and UIFprovides mRNA-adaptor redundancy in Herpesvirus intronless mRNA export. PLoS Pathog. 7, e1002138 doi:10.1371/journal.ppat.1002138

187 Koffa, M.D., Clements, J.B., Izaurralde, E., Wadd, S., Wilson, S.A., Mattaj, I.W. et al. (2001) Herpes simplex virus ICP27 protein provides viral mRNAswith access to the cellular mRNA export pathway. EMBO J. 20, 5769–5778 doi:10.1093/emboj/20.20.5769

188 Hiriart, E., Farjot, G., Gruffat, H., Nguyen, M.V., Sergeant, A. and Manet, E. (2002) A novel nuclear export signal and a REF interaction domain bothpromote mRNA export by the Epstein-Barr virus EB2 protein. J. Biol. Chem. 278, 335–342 doi:10.1074/jbc.M208656200

189 Lischka, P., Toth, Z., Thomas, M., Mueller, R. and Stamminger, T. (2006) The UL69 transactivator protein of human cytomegalovirus interactswith DEXD/H-Box RNA helicase UAP56 to promote cytoplasmic accumulation of unspliced RNA. Mol. Cell. Biol. 26, 1631–1643doi:10.1128/MCB.26.5.1631-1643.2006

190 Balasubramaniam, V.R., Hong Wai, T., Ario Tejo, B., Omar, A.R. and Syed Hassan, S. (2013) Highly pathogenic avian influenza virus nucleoproteininteracts with TREX complex adaptor protein Aly/REF. PLoS ONE 8, e72429 doi:10.1371/journal.pone.0072429

191 Stubbs, S.H., Hunter, O.V., Hoover, A. and Conrad, N.K. (2012) Viral factors reveal a role for REF/Aly in nuclear RNA stability. Mol. Cell Biol. 32,1260–1270 doi:10.1128/MCB.06420-11

192 Kammel, C., Thomaier, M., Sørensen, B.B., Schubert, T., Längst, G., Grasser, M. et al. (2013) Arabidopsis DEAD-box RNA helicase UAP56 interactswith both RNA and DNA as well as with mRNA export factors. PLoS ONE 8, e60644 doi:10.1371/journal.pone.0060644

193 Pan, H., Liu, S. and Tang, D. (2012) HPR1, a component of the THO/TREX complex, plays an important role in disease resistance and senescence inArabidopsis. Plant J. 69, 831–843 doi:10.1111/j.1365-313X.2011.04835.x

194 Jauvion, V., Elmayan, T. and Vaucheret, H. (2010) The conserved RNA trafficking proteins HPR1 and TEX1 are involved in the production of endogenousand exogenous small interfering RNA in Arabidopsis. Plant Cell. 22, 2697–2709 doi:10.1105/tpc.110.076638

195 Xu, C., Zhou, X. and Wen, C.K. (2015) HYPER RECOMBINATION1 of the THO/TREX complex plays a role in controlling transcription of theREVERSION-TO-ETHYLENE SENSITIVITY1 gene in Arabidopsis. PLoS Genet. 11, e1004956 doi:10.1371/journal.pgen.1004956

196 Pitzonka, L., Wang, X., Ullas, S., Wolff, D.W., Wang, Y. and Goodrich, D.W. (2013) The THO ribonucleoprotein complex is required for stem cellhomeostasis in the adult mouse small intestine. Mol. Cell. Biol. 33, 3505–3514

197 Isomura, M., Okui, K., Fujiwara, T., Shin, S. and Nakamura, Y. (1996) Cloning and mapping of a novel human cDNA homologous to DROER, theenhancer of the Drosophila melanogaster rudimentary gene. Genomics 32, 125–127

198 Black, B.E., Levesque, L., Holaska, J.M., Wood, T.C. and Paschal, B.M. (1999) Identification of an NTF2-related factor that binds Ran-GTP and regulatesnuclear protein export. Mol. Cell. Biol. 19, 8616–8624

199 Guzik, B.W., Levesque, L., Prasad, S., Bor, Y.C., Black, B.E., Paschal, B.M. et al. (2001) NXT1 (p15) is a crucial cellular cofactor in TAP-dependentexport of intron-containing RNA in mammalian cells. Mol. Cell. Biol. 21, 2545–2554

200 Lubas, M., Christensen, M.S., Kristiansen, M.S., Domanski, M., Falkenby, L.G., Lykke-Andersen, S. et al. (2011) Interaction profiling identifies thehuman nuclear exosome targeting complex. Mol. Cell. 43, 624–637

201 Wilson, M.D., Wang, D., Wagner, R., Breyssens, H., Gertsenstein, M., Lobe, C. et al. (2008) ARS2 is a conserved eukaryotic gene essential for earlymammalian development. Mol. Cell. Biol. 28, 1503–1514

202 Gruber, J.J., Zatechka, D.S., Sabin, L.R., Yong, J., Lum, J.J., Kong, M. et al. (2009) Ars2 links the nuclear cap-binding complex to RNA interferenceand cell proliferation. Cell 138, 328–339

203 Hallais, M., Pontvianne, F., Andersen, P.R., Clerici, M., Lener, D., Benbahouche, N.H. et al. (2013) CBC-ARS2 stimulates 3’-end maturation of multipleRNA families and favors cap-proximal processing. Nat. Struct. Mol. Biol. 20, 1358–1366

204 Merz, C., Urlaub, H., Will, C.L. and Lührmann, R. (2007) Protein composition of human mRNPs spliced in vitro and differential requirements for mRNPprotein recruitment. RNA 13, 116–128

205 Izaurralde, E., Lewis, J., McGuigan, C., Jankowska, M., Darzynkiewicz, E. and Mattaj, I.W. (1994) A nuclear cap binding protein complex involved in pre-mRNA splicing. Cell 78, 657–668

© 2016 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY) 2935

Biochemical Journal (2016) 473 2911–2935DOI: 10.1042/BCJ20160010